Protein

ABSTRACT

The present invention provides methods and compositions for the treatment of and for screening, diagnosis and prognosis of colorectal cancer, for monitoring the effectiveness of colorectal cancer treatment, and for drug development.

RELATED APPLICATIONS

The present application is a Continuation-In-Part of co-pending U.S.Non-Provisional application Ser. No. 12/395,569, filed Feb. 27, 2009,which in turn, is a continuation of PCT Application No.PCT/GB2007/050513 filed Aug. 29, 2007, which in turn, claims priorityfrom G.B. Application No. 0616971.8 filed Aug. 29, 2006 and U.S.Provisional Application Ser. No. 60/842,431 filed Sep. 6, 2006.

The present application is also a Continuation-In-Part of co-pendingU.S. Non-Provisional application Ser. No. 12/329,500, filed Dec. 5,2008, which in turn, is a continuation of PCT Application No.PCT/EP2007/055537 filed Jun. 5, 2007, which in turn, claims priorityfrom G.B. Application No. 0611116.5 filed Jun. 6, 2006 and U.S.Provisional Application Ser. No. 60/811,681 filed Jun. 7, 2006.

The present application is also a Continuation-In-Part of co-pending PCTApplication No. PCT/US2010/031719, filed Apr. 20, 2010, which in turn,claims priority from U.S. Provisional Application Ser. No. 61/170,980,filed Apr. 20, 2009.

Applicants claim the benefits of 35 U.S.C. §120 as to the U.S.Non-Provisional Applications and the PCT applications, and priorityunder 35 U.S.C. §119 as to the said G.B. and U.S. Provisionalapplications, and the entire disclosures of all applications areincorporated herein by reference in their entireties.

INTRODUCTION

The present invention relates to the identification of a membraneprotein associated with colorectal cancer which has utility as a targetfor the treatment of colorectal cancer, in particular using antibodieswhich bind to said membrane protein, and has utility as amarker forcolorectal cancer and colorectal cancer metastases and which also formsa biological target against which therapeutic antibodies (or otheraffinity reagents such as Affibodies, Nanobodies or Unibodies) or otherpharmaceutical agents can be made.

BACKGROUND OF THE INVENTION Colorectal Cancer:

Colorectal cancer (CRC) is one of the leading causes of cancer-relatedmorbidity and mortality, responsible for an estimated half a milliondeaths per year, mostly in Western, well developed countries. In theseterritories, CRC is the third most common malignancy (estimated numberof new cases per annum in USA and EU is approximately 350,000 per year).Estimated healthcare costs related to treatment for colorectal cancer inthe United States are more than $8 billion.

Colorectal Cancer Diagnosis:

Today, the fecal occult blood test and colonoscopy, a highly invasiveprocedure, are the most frequently used screening and diagnostic methodsfor colorectal cancer.

Other diagnostic tools include Flexible Sigmoidoscopy (allowing theobservation of only about half of the colon) and Double Contrast BariumEnema (DCBE, to obtain X-ray images).

Colorectal Cancer Staging:

CRC has four distinct stages: patients with stage I disease, have afive-year survival rate of >90%, while those with metastatic stage IVdisease have a <5% survival rate according to the US National Institutesof Health (NIH).

Colorectal Cancer Treatment:

Once CRC has been diagnosed, the correct treatment needs to be selected.Surgery is usually the main treatment for rectal cancer, althoughradiation and chemotherapy will often be given before surgery. Possibleside effects of surgery include bleeding from the surgery, deep veinthrombosis, and damage to nearby organs during the operation.

Currently, 60 percent of colorectal cancer patients receive chemotherapyto treat their disease; however, this form of treatment only benefits afew percent of the population, while carrying with it high risks oftoxicity, thus demonstrating a need to better define the patientselection criteria.

Colorectal cancer has a 30 to 40 percent recurrence rate within anaverage of 18 months after primary diagnosis. As with all cancers, theearlier it is detected the more likely it can be cured, especially aspathologists have recognised that the majority of CRC tumours develop ina series of well-defined stages from benign adenomas.

Colon Cancer Survival by Stage

Stage Survival Rate I 93% IIA 85% IIB 72% IIIA 83% IIIB 64% IIIC 44% IV 8%

Therapeutic Challenges

The major challenges in colorectal cancer treatment are to improve earlydetection rates, to find new non-invasive markers that can be used tofollow disease progression and identify relapse, and to find improvedand less toxic therapies, especially for more advanced disease where 5year survival is still very poor. There is a great need to identifytargets which are more specific to the cancer cells, e.g. ones which areexpressed on the surface of the tumour cells so that they can beattacked by promising new approaches like immunotherapeutics andtargeted toxins.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for screening,diagnosis, prognosis and therapy of colorectal cancer, for colorectalcancer patients' stratification, for monitoring the effectiveness ofcolorectal cancer treatment, and for drug development for treatment ofcolorectal cancer.

We have used mass spectrometry to identify peptides generated by gelelectrophoresis or tagging with iTRAQ reagents and tryptic digest ofmembrane proteins extracted from colorectal cancer tissue samples.Peptide sequences were compared to existing protein and cDNA databasesand the corresponding gene sequences identified. The protein of theinvention has not been previously reported to originate from colorectalcancer cell membranes and represents a protein of new diagnostic andtherapeutic value.

Thus, a first aspect of the invention provides methods for diagnosis ofcolorectal cancer that comprises analysing a sample of colon tissue e.g.by gel electrophoresis, iTRAQ or other appropriate protein separationtechnique to detect the protein of the invention. Such methods are alsoset forth in commonly assigned, co-pending parent application Ser. No.12/329,500, filed Dec. 5, 2008, the disclosure of which is incorporatedherein in its entirety. These methods are also suitable for screening,prognosis, monitoring the results of therapy, drug development anddiscovery of new targets for drug treatment.

A second aspect of the invention provides methods of treating colorectalcancer, comprising administering to a patient a therapeuticallyeffective amount of a compound that modulates (e.g., upregulates ordownregulates) or complements the expression or the biological activity(or both) of the protein of the invention in patients having colorectalcancer, in order to (a) prevent the onset or development of colorectalcancer; (b) prevent the progression of colorectal cancer; or (c)ameliorate the symptoms of colorectal cancer.

A third aspect of the invention provides methods of screening forcompounds that modulate (e.g., upregulate or downregulate) theexpression or biological activity of the protein of the invention.

A fourth aspect of the invention provides monoclonal and polyclonalantibodies or other affinity reagents such as Affibodies, Nanobodies orUnibodies capable of immunospecific binding to the protein of theinvention.

Thus, in a fifth aspect, the present invention provides a method forscreening for and/or diagnosis of colorectal cancer in a human subject,which method comprises the step of identifying the presence or absenceof the protein of the invention, in a biological sample obtained fromsaid human subject.

In a sixth aspect, the present invention provides a method formonitoring and/or assessing colorectal cancer treatment in a humansubject, which comprises the step of identifying the presence or absenceof the protein of the invention, in a biological sample obtained fromsaid human subject.

In a seventh aspect, the present invention provides a method foridentifying the presence or absence of metastatic colorectal cancercells in a biological sample obtained from a human subject, whichcomprises the step of identifying the presence or absence of the proteinof the invention.

In an eighth aspect, the present invention provides a method formonitoring and/or assessing colorectal cancer treatment in a humansubject, which comprises the step of determining whether the protein ofthe invention is increased/decreased in a biological sample obtainedfrom a patient.

The biological sample used can be from any source such as a serum sampleor a tissue sample, e.g. colorectal tissue. For instance, when lookingfor evidence of metastatic colorectal cancer, one would look at majorsites of colorectal cancer metastasis, e.g. the liver, the peritonealcavity, the pelvis, the retroperitoneum and the lungs.

Other aspects of the present invention are set out below and in theclaims herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence of the protein of the inventionisolated from colorectal cancer samples using 1D-gel electrophoresistechnology. The tryptic fragments detected experimentally by massspectrometry are highlighted, mass match peptides are shown in bold andtandem peptides are underlined.

FIG. 2 is similar to FIG. 1, except the protein of the invention wasisolated from colorectal cancer samples using iTRAQ.

FIGS. 3 a-3 i-show the Protein Index for the protein of the invention invarious diseases. The list of diseases investigated is shown in Table 3awith Table 3b indicating subcellular locations.

FIG. 4 shows ROC curve data for the presence of the protein of theinvention in colorectal cancer patient serum samples.

FIG. 5 shows results of FACS analysis on CDH17_A4 in LoVo and LS174Tcells.

FIG. 6A shows results of internalisation of CDH17_A4 by MabZAP assay inLoVo colon cancer cells.

FIG. 6B shows results of internalisation of CDH17_A4 by MabZAP assay inLoVo colon cancer cells.

FIG. 6C shows results of internalisation of CDH17_A4 by MabZAP assay inLS174T colon cancer cells.

FIG. 6D shows results of internalisation of CDH17_A4 by MabZAP assay inLS174T colon cancer cells.

FIG. 7A shows results of internalisation of CDH17_A4 by MabZAP assay inLoVo colon cancer cells.

FIG. 7B shows results of internalisation of CDH17_A4 by MabZAP assay inLS174T colon cancer cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention described in detail below provides methods andcompositions for clinical screening, diagnosis and prognosis ofcolorectal cancer in a mammalian subject for identifying patients mostlikely to respond to a particular therapeutic treatment, for monitoringthe results of colorectal cancer therapy, for drug screening and drugdevelopment. The invention also encompasses the administration oftherapeutic compositions to a mammalian subject to treat or preventcolorectal cancer. The mammalian subject may be a non-human mammal, butis preferably human, more preferably a human adult, i.e. a human subjectat least 21 (more preferably at least 35, at least 50, at least 60, atleast 70, or at least 80) years old. For clarity of disclosure, and notby way of limitation, the invention will be described with respect tothe analysis of colon tissue. However, as one skilled in the art willappreciate, the assays and techniques described below can be applied toother types of patient samples, including body fluids (e.g. blood, urineor saliva), a tissue sample from a patient at risk of having colorectalcancer (e.g. a biopsy such as a colorectal biopsy) or homogenatethereof. The methods and compositions of the present invention arespecially suited for screening, diagnosis and prognosis of a livingsubject, but may also be used for postmortem diagnosis in a subject, forexample, to identify family members at risk of developing the samedisease.

As used herein, colon tissue refers to the colon itself, as well as thetissue adjacent to and/or within the strata underlying the colon.

OGTA001

In one aspect of the invention, one-dimensional electrophoresis orisobaric tags for relative and absolute quantification (iTRAQ) are usedto analyze colorectal cancer tissue samples from a subject, preferably aliving subject, in order to measure the expression of the protein of theinvention for screening or diagnosis of colorectal cancer, to determinethe prognosis of a colorectal cancer patient, to monitor theeffectiveness of colorectal cancer therapy, or for drug development.

As used herein, the term “Protein of the invention”, or “OGTA001”,refers to the protein illustrated in FIG. 1 detected experimentally by1D gel electrophoresis and iTRAQ analysis of colorectal tissue samples.Protein derivatives of this sequence may also be useful for the samepurposes as described herein.

This protein has been identified in membrane protein extracts ofcolorectal tissue samples from colorectal cancer patients, through themethods and apparatus of the Preferred Technologies (1D gelelectrophoresis or iTRAQ together with tryptic digest of membraneprotein extracts). Peptide sequences were compared to the SWISS-PROT andtrEMBL databases (held by the Swiss Institute of Bioinformatics (SIB)and the European Bioinformatics Institute (EBI) which are available atwww.expasy.com), and the following entry: Q12864, Cadherin-17, wasidentified.

According to SWISS-PROT, Cadherin-17 is known to be predominantlyexpressed in the gastrointestinal tract and pancreatic duct. It is notdetected in kidney, lung, liver, brain, adrenal gland or skin. Cadherinsare calcium dependent cell adhesion proteins. They preferentiallyinteract with themselves in a homophilic manner in connecting cells;cadherins may thus contribute to the sorting of heterogeneous celltypes. Cadherin-17 may have a role in the morphological organization ofliver and intestine. It is involved in intestinal peptide transport.

The protein of the invention is useful as are fragments e.g. antigenicor immunogenic fragments thereof and derivatives thereof. Antigenic orimmunogenic fragments will typically be of length 12 amino acids or moree.g. 20 amino acids or more e.g. 50 or 100 amino acids or more.Fragments may be 10% or more of the length of the full protein e.g. 25%or more e.g. 50% or 75% or 90% or 95% or more of the length of the fullprotein.

Antigenic or immunogenic fragments will be capable of eliciting arelevant immune response in a patient. DNA encoding the protein of theinvention is also useful as are fragments thereof e.g. DNA encodingfragments of the protein of the invention such as immunogenic fragmentsthereof. Fragments of nucleic acid (e.g. DNA) encoding the protein ofthe invention may be 10% or more of the length of the full coding regione.g. 25% or more e.g. 50% or 75% or 90% or 95% or more of the length ofthe full coding region. Fragments of nucleic acid (e.g. DNA) may be 36nucleotides or more e.g. 60 nucleotides or more e.g. 150 or 300nucleotides or more in length.

Derivatives of the protein of the invention include variants on thesequence in which one or more (e.g. 1-20 such as 15 amino acids, or upto 20% such as up to 10% or 5% or 1% by number of amino acids based onthe total length of the protein) deletions, insertions or substitutionshave been made. Substitutions may typically be conservativesubstitutions. For example derivatives may have sequence identity of 80%or more e.g. 90% or more e.g. 95% or more as compared with the referencesequence over the full length of the reference sequence. Derivativeswill typically have essentially the same biological function as theprotein from which they are derived. Derivatives will typically becomparably antigenic or immunogenic to the protein from which they arederived.

Table 1 below illustrates the different occurrences of OGTA001 asdetected by 1D gel electrophoresis and mass spectrometry of membraneprotein extracts of colorectal tissue samples from colorectal cancerpatients. The first column provides the molecular weight, the secondcolumn gives information on the subfractionation protocol used, if any(see Example 1 below), and the last column provides a list of thesequences observed by mass spectrometry and the corresponding SEQ IDNos.

Table 2 below illustrates the different occurrences of OGTA001 asdetected by iTRAQ and mass spectrometry of membrane protein extracts ofcolorectal tissue samples from colorectal cancer patients. The firstcolumn provides the sample number, the second column gives informationon the iTRAQ experiment number for that sample and the last columnprovides a list of the sequences observed by mass spectrometry and thecorresponding SEQ ID Nos.

TABLE 1 Colorectal cancer 1D gel MW Subfraction- (Da) ationTryptics identified [SEQ ID No] 91099DEENTANSFLNYR [4], DNVESAQASEVKPLR [7],  WNDPGAQYSLVDK [13] 114588Nucleotide  AENPEPLVFGVK [2], DAYVFYAVAK [3],  BindingDEENTANSFLNYR [4], DINDNRPTFLQSK [6], DNVESAQASEVKPLR [7] 117144Nucleotide  DEENTANSFLNYR [4], DEYGKPLSYPLEIHVK [5],  BindingDNVESAQASEVKPLR [7], IDHVTGEIFSVAPLDR [10] 119837 Nucleotide AENPEPLVFGVK [2], DEENTANSFLNYR [4],  BindingDNVESAQASEVKPLR [7], TGAISLTR [13] 125678 Nucleotide DEENTANSFLNYR [4], DNVESAQASEVKPLR [7] Binding

TABLE 2 Colorectal cancer iTRAQ Sample Experiment no. no.Tryptics identified [SEQ ID No] Sample 1 Experiment 1AENPEPLVFGVK [2], DEYGKPLSYPLEIHVK [5], GWLK [9],IDHVTGEIFSVAPLDR [10], KPLDFETAAVSNIVFK [11],LGVDTDPHTNTGYVIIK [12], VKDINDNPPTCPSPVTVFEVQENER[14], VSEDVAIGTK [15], WNDPGAQYSLVDKEKLPR [17] Sample 1 Experiment 2AENPEPLVFGVK [2], IDHVTGEIFSVAPLDR [10] Sample 2 Experiment 1EGSQELNPAK [8]

For OGTA001, the detected level obtained upon analyzing tissue fromsubjects having colorectal cancer relative to the detected levelobtained upon analyzing tissue from subjects free from colorectal cancerwill depend upon the particular analytical protocol and detectiontechnique that is used. Accordingly, the present invention contemplatesthat each laboratory will establish a reference range in subjects freefrom colorectal cancer according to the analytical protocol anddetection technique in use, as is conventional in the diagnostic art.Preferably, at least one control positive tissue sample from a subjectknown to have colorectal cancer or at least one control negative tissuesample from a subject known to be free from colorectal cancer (and morepreferably both positive and negative control samples) are included ineach batch of test samples analysed.

OGTA001 can be used for detection, prognosis, diagnosis, or monitoringof colorectal cancer or for drug development. In one embodiment of theinvention, tissue from a subject (e.g., a subject suspected of havingcolorectal cancer) is analysed by 1D electrophoresis or iTRAQ fordetection of OGTA001. An increased abundance of OGTA001 in the tissuefrom the subject relative to tissue from a subject or subjects free fromcolorectal cancer (e.g., a control sample) or a previously determinedreference range indicates the presence of colorectal cancer.

In relation to fragments, immunogenic fragments or antigenic fragmentsof OGTA001, for colorectal cancer applications, preferably thesecomprise one or more of the sequences identified as tryptic sequences inthe 3^(rd) column of Table 1 or the 3^(rd) column of Table 2.

OGTA001 may, in particular, be characterized as an isoform having a MWsubstantially as recited (eg +/−10%, particularly +/−5% of the value) incolumn 1 of any of the rows of Table 1.

The present invention additionally provides: (a) a preparationcomprising isolated OGTA001; (b) a preparation comprising one or morefragments of OGTA001; and (c) antibodies or other affinity reagents suchas Affibodies, Nanobodies or Unibodies that bind to OGTA001, to saidfragments, or both to OGTA001 and to said fragments. As used herein,OGTA001 is “isolated” when it is present in a preparation that issubstantially free of contaminating proteins, i.e., a preparation inwhich less than 10% (preferably less than 5%, more preferably less than1%) of the total protein present is contaminating protein(s). Acontaminating protein is a protein having a significantly differentamino acid sequence from that of isolated OGTA001, as determined by massspectral analysis. As used herein, a “significantly different” sequenceis one that permits the contaminating protein to be resolved fromOGTA001 by mass spectral analysis, performed according to the ReferenceProtocols.

OGTA001 can be assayed by any method known to those skilled in the art,including but not limited to, the Preferred Technologies describedherein, kinase assays, enzyme assays, binding assays and otherfunctional assays, immunoassays, and western blotting. In oneembodiment, OGTA001 is separated on a 1-D gel by virtue of its MW andvisualized by staining the gel. In one embodiment, OGTA001 is stainedwith a fluorescent dye and imaged with a fluorescence scanner. Sypro Red(Molecular Probes, Inc., Eugene, Oreg.) is a suitable dye for thispurpose. A preferred fluorescent dye is disclosed in U.S. applicationSer. No. 09/412,168, filed on Oct. 5, 1999, which is incorporated hereinby reference in its entirety. In another embodiment, OGTA001 is analysedusing isobaric tags for relative and absolute quantification (iTRAQ).

Alternatively, OGTA001 can be detected in an immunoassay. In oneembodiment, an immunoassay is performed by contacting a sample from asubject to be tested with an anti-OGTA001 antibody (or other affinityreagent such as an Affibody, Nanobody or Unibody) under conditions suchthat immunospecific binding can occur if OGTA001 is present, anddetecting or measuring the amount of any immunospecific binding by theaffinity reagent. Anti-OGTA001 affinity reagents can be produced by themethods and techniques taught herein.

OGTA001 may be detected by virtue of the detection of a fragment thereofe.g. an immunogenic or antigenic fragment thereof. Fragments may have alength of at least 10, more typically at least 20 amino acids eg atleast 50 or 100 amino acids eg at least 200 or 500 amino acids.

In one embodiment, binding of antibody (or other affinity reagent suchas an Affibody, Nanobody or Unibody) in tissue sections can be used todetect aberrant OGTA001 localization or an aberrant level of OGTA001. Ina specific embodiment, an antibody (or other affinity reagent such as anAffibody, Nanobody or Unibody) to OGTA001 can be used to assay a patienttissue (e.g., a colon tissue) for the level of OGTA001 where an aberrantlevel of OGTA001 is indicative of colorectal cancer. As used herein, an“aberrant level” means a level that is increased compared with the levelin a subject free from colorectal cancer or a reference level.

Any suitable immunoassay can be used, including, without limitation,competitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays and protein A immunoassays.

For example, OGTA001 can be detected in a fluid sample (e.g., blood,urine, or saliva) by means of a two-step sandwich assay. In the firststep, a capture reagent (e.g., an anti-OGTA001 antibody or otheraffinity reagent such as an Affibody, Nanobody or Unibody) is used tocapture OGTA001. The capture reagent can optionally be immobilized on asolid phase. In the second step, a directly or indirectly labeleddetection reagent is used to detect the captured OGTA001. In oneembodiment, the detection reagent is a lectin. Any lectin can be usedfor this purpose that preferentially binds to OGTA001 rather than toother isoforms that have the same core protein as OGTA001 or to otherproteins that share the antigenic determinant recognized by the affinityreagent. In a preferred embodiment, the chosen lectin binds OGTA001 withat least 2-fold greater affinity, more preferably at least 5-foldgreater affinity, still more preferably at least 10-fold greateraffinity, than to said other isoforms that have the same core protein asOGTA001 or to said other proteins that share the antigenic determinantrecognized by the affinity reagent. Based on the present description, alectin that is suitable for detecting OGTA001 can readily be identifiedby methods well known in the art, for instance upon testing one or morelectins enumerated in Table I on pages 158-159 of Sumar et al., Lectinsas Indicators of Disease-Associated Glycoforms, In: Gabius H-J & GabiusS (eds.), 1993, Lectins and Glycobiology, at pp. 158-174 (which isincorporated herein by reference in its entirety). In an alternativeembodiment, the detection reagent is an antibody (or other affinityreagent such as an Affibody, Nanobody or Unibody), e.g., an antibodythat immunospecifically detects other post-translational modifications,such as an antibody that immunospecifically binds to phosphorylatedamino acids. Examples of such antibodies include those that bind tophosphotyrosine (BD Transduction Laboratories, catalog nos.:P11230-050/P11230-150; P11120; P38820; P39020), those that bind tophosphoserine (Zymed Laboratories Inc., South San Francisco, Calif.,catalog no. 61-8100) and those that bind to phosphothreonine (ZymedLaboratories Inc., South San Francisco, Calif., catalogue nos. 71-8200,13-9200).

If desired, a gene encoding OGTA001, a related gene, or related nucleicacid sequences or subsequences, including complementary sequences, canalso be used in hybridization assays. A nucleotide encoding OGTA001, orsubsequences thereof comprising at least 8 nucleotides, preferably atleast 12 nucleotides, and most preferably at least 15 nucleotides can beused as a hybridization probe. Hybridization assays can be used fordetection, prognosis, diagnosis, or monitoring of conditions, disorders,or disease states, associated with aberrant expression of the geneencoding OGTA001, or for differential diagnosis of subjects with signsor symptoms suggestive of colorectal cancer. In particular, such ahybridization assay can be carried out by a method comprising contactinga subject's sample containing nucleic acid with a nucleic acid probecapable of hybridizing to a DNA or RNA that encodes OGTA001, underconditions such that hybridization can occur, and detecting or measuringany resulting hybridization.

The invention also provides diagnostic kits, comprising an anti-OGTA001antibody (or other affinity reagent such as an Affibody, Nanobody orUnibody). In addition, such a kit may optionally comprise one or more ofthe following: (1) instructions for using the anti-OGTA001 affinityreagent for diagnosis, prognosis, therapeutic monitoring or anycombination of these applications; (2) a labeled binding partner to theaffinity reagent; (3) a solid phase (such as a reagent strip) upon whichthe anti-OGTA001 affinity reagent is immobilized; and (4) a label orinsert indicating regulatory approval for diagnostic, prognostic ortherapeutic use or any combination thereof. If no labeled bindingpartner to the affinity reagent is provided, the anti-OGTA001 affinityreagent itself can be labeled with a detectable marker, e.g., achemiluminescent, enzymatic, fluorescent, or radioactive moiety.

The invention also provides a kit comprising a nucleic acid probecapable of hybridizing to RNA encoding OGTA001. In a specificembodiment, a kit comprises in one or more containers a pair of primers(e.g., each in the size range of 6-30 nucleotides, more preferably 10-30nucleotides and still more preferably 10-20 nucleotides) that underappropriate reaction conditions can prime amplification of at least aportion of a nucleic acid encoding OGTA001, such as by polymerase chainreaction (see, e.g., Innis et al., 1990, PCR Protocols, Academic Press,Inc., San Diego, Calif.), ligase chain reaction (see EP 320,308) use ofQ13 replicase, cyclic probe reaction, or other methods known in the art.

A kit can optionally further comprise a predetermined amount of OGTA001or a nucleic acid encoding OGTA001, e.g., for use as a standard orcontrol.

Use in Clinical Studies

The diagnostic methods and compositions of the present invention canassist in monitoring a clinical study, e.g. to evaluate drugs fortherapy of colorectal cancer. In one embodiment, candidate molecules aretested for their ability to restore OGTA001 levels in a subject havingcolorectal cancer to levels found in subjects free from colorectalcancer or, in a treated subject, to preserve OGTA001 levels at or nearnon-colorectal cancer values.

In another embodiment, the methods and compositions of the presentinvention are used to screen candidates for a clinical study to identifyindividuals having colorectal cancer; such individuals can then beexcluded from the study or can be placed in a separate cohort fortreatment or analysis.

Production of Protein of the Invention and Corresponding Nucleic Acid

A DNA of the present invention can be obtained by isolation as a cDNAfragment from cDNA libraries using as starter materials commercial mRNAsand determining and identifying the nucleotide sequences thereof. Thatis, specifically, clones are randomly isolated from cDNA libraries,which are prepared according to Ohara et al's method (DNA Research Vol.4, 53-59 (1997)). Next, through hybridization, duplicated clones (whichappear repeatedly) are removed and then in vitro transcription andtranslation are carried out. Nucleotide sequences of both termini ofclones, for which products of 50 kDa or more are confirmed, aredetermined. Furthermore, databases of known genes are searched forhomology using the thus obtained terminal nucleotide sequences asqueries. The entire nucleotide sequence of a clone revealed to be novelas a result is determined. In addition to the above screening method,the 5′ and 3′ terminal sequences of cDNA are related to a human genomesequence. Then an unknown long-chain gene is confirmed in a regionbetween the sequences, and the full-length of the cDNA is analyzed. Inthis way, an unknown gene that is unable to be obtained by aconventional cloning method that depends on known genes can besystematically cloned.

Moreover, all of the regions of a human-derived gene containing a DNA ofthe present invention can also be prepared using a PCR method such asRACE while paying sufficient attention to prevent artificial errors fromtaking place in short fragments or obtained sequences. As describedabove, clones having DNA of the present invention can be obtained.

In another means for cloning DNA of the present invention, a syntheticDNA primer having an appropriate nucleotide sequence of a portion of apolypeptide of the present invention is produced, followed byamplification by the PCR method using an appropriate library.Alternatively, selection can be carried out by hybridization of the DNAof the present invention with a DNA that has been incorporated into anappropriate vector and labeled with a DNA fragment or a synthetic DNAencoding some or all of the regions of the polypeptide of the presentinvention. Hybridization can be carried out by, for example, the methoddescribed in Current Protocols in Molecular Biology (edited by FrederickM. Ausubel et al., 1987). DNA of the present invention may be any DNA,as long as they contain nucleotide sequences encoding the polypeptidesof the present invention as described above. Such a DNA may be a cDNAidentified and isolated from cDNA libraries or the like that are derivedfrom colorectal tissue. Such a DNA may also be a synthetic DNA or thelike. Vectors for use in library construction may be any ofbacteriophages, plasmids, cosmids, phargemids, or the like. Furthermore,by the use of a total RNA fraction or a mRNA fraction prepared from theabove cells and/or tissues, amplification can be carried out by a directreverse transcription coupled polymerase chain reaction (hereinafterabbreviated as “RT-PCR method”).

DNA encoding the above polypeptide consisting of an amino acid sequencethat is substantially identical to the amino acid sequence of OGTA001 orDNA encoding the above polypeptide consisting of an amino acid sequencederived from the amino acid sequence of OGTA001 by deletion,substitution, or addition of one or more amino acids composing a portionof the amino acid sequence can be easily produced by an appropriatecombination of, for example, a site-directed mutagenesis method, a genehomologous recombination method, a primer elongation method, and the PCRmethod known by persons skilled in the art. In addition, at this time, apossible method for causing a polypeptide to have substantiallyequivalent biological activity is substitution of homologous amino acids(e.g. polar and nonpolar amino acids, hydrophobic and hydrophilic aminoacids, positively-charged and negatively charged amino acids, andaromatic amino acids) among amino acids composing the polypeptide.Furthermore, to maintain substantially equivalent biological activity,amino acids within functional domains contained in the polypeptide ofthe present invention are preferably conserved.

Furthermore, examples of DNA of the present invention include DNAcomprising a nucleotide sequence that encodes the amino acid sequence ofOGTA001 and DNA hybridizing under stringent conditions to the DNA andencoding a polypeptide (protein) having biological activity (function)equivalent to the function of the polypeptide consisting of the aminoacid sequence of OGTA001. Under such conditions, an example of such DNAcapable of hybridizing to DNA comprising the nucleotide sequence thatencodes the amino acid sequence of OGTA001 is DNA comprising anucleotide sequence that has a degree of overall mean homology with theentire nucleotide sequence of the DNA, such as approximately 80% ormore, preferably approximately 90% or more, and more preferablyapproximately 95% or more. Hybridization can be carried out according toa method known in the art such as a method described in CurrentProtocols in Molecular Biology (edited by Frederick M. Ausubel et al.,1987) or a method according thereto. Here, “stringent conditions” are,for example, conditions of approximately “1*SSC, 0.1% SDS, and 37° C.,more stringent conditions of approximately “0.5*SSC, 0.1% SDS, and 42°C., or even more stringent conditions of approximately “0.2*SSC, 0.1%SDS, and 65° C. With more stringent hybridization conditions, theisolation of a DNA having high homology with a probe sequence can beexpected. The above combinations of SSC, SDS, and temperature conditionsare given for illustrative purposes. Stringency similar to the above canbe achieved by persons skilled in the art using an appropriatecombination of the above factors or other factors (for example, probeconcentration, probe length, and reaction time for hybridization) fordetermination of hybridization stringency.

A cloned DNA of the present invention can be directly used or used, ifdesired, after digestion with a restriction enzyme or addition of alinker, depending on purposes. The DNA may have ATG as a translationinitiation codon at the 5′ terminal side and have TAA, TGA, or TAG as atranslation termination codon at the 3′ terminal side. These translationinitiation and translation termination codons can also be added using anappropriate synthetic DNA adapter.

Where it is provided for use with the methods of the invention OGTA001is preferably provided in isolated form. More preferably the OGTA001polypeptide has been purified to at least to some extent. OGTA001polypeptide may be provided in substantially pure form, that is to sayfree, to a substantial extent, from other proteins. OGTA001 polypeptidecan also be produced using recombinant methods, synthetically producedor produced by a combination of these methods. OGTA001 can be easilyprepared by any method known by persons skilled in the art, whichinvolves producing an expression vector containing a DNA of the presentinvention or a gene containing a DNA of the present invention, culturinga transformant transformed using the expression vector, generating andaccumulating a polypeptide of the present invention or a recombinantprotein containing the polypeptide, and then collecting the resultant.

Recombinant OGTA001 polypeptide may be prepared by processes well knownin the art from genetically engineered host cells comprising expressionsystems. Accordingly, the present invention also relates to expressionsystems which comprise an OGTA001 polypeptide or nucleic acid, to hostcells which are genetically engineered with such expression systems andto the production of OGTA001 polypeptide by recombinant techniques. Forrecombinant OGTA001 polypeptide production, host cells can begenetically engineered to incorporate expression systems or portionsthereof for nucleic acids. Such incorporation can be performed usingmethods well known in the art, such as, calcium phosphate transfection,DEAD-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction or infection (see e.g. Davis etal., Basic Methods in Molecular Biology, 1986 and Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbourlaboratory Press, Cold Spring Harbour, N.Y., 1989).

As host cells, for example, bacteria of the genus Escherichia,Streptococci, Staphylococci, Streptomyces, bacteria of the genusBacillus, yeast, Aspergillus cells, insect cells, insects, and animalcells are used. Specific examples of bacteria of the genus Escherichia,which are used herein, include Escherichia coli K12 and DH1 (Proc. Natl.Acad. Sci. U.S.A., Vol. 60, 160 (1968)), JM103 (Nucleic Acids Research,Vol. 9, 309 (1981)), JA221 (Journal of Molecular Biology, Vol. 120, 517(1978)), and HB101 (Journal of Molecular Biology, Vol. 41, 459 (1969)).As bacteria of the genus Bacillus, for example, Bacillus subtilis MI114(Gene, Vol. 24, 255 (1983)) and 207-21 (Journal of Biochemistry, Vol.95, 87 (1984)) are used. As yeast, for example, Saccaromyces cerevisiaeAH22, AH22R-, NA87-11A, DKD-5D, and 20B-12, Schizosaccaromyces pombeNCYC1913 and NCYC2036, and Pichia pastoris are used. As insect cells,for example, Drosophila S2 and Spodoptera Sf9 cells are used. As animalcells, for example, COS-7 and Vero monkey cells, CHO Chinese hamstercells (hereinafter abbreviated as CHO cells), dhfr-gene-deficient CHOcells, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3cells, human FL cells, COS, HeLa, C127,3T3, HEK 293, BHK and Bowesmelanoma cells are used.

Cell-free translation systems can also be employed to producerecombinant polypeptides (e.g. rabbit reticulocyte lysate, wheat germlysate, SP6/T7 in vitro T&T and RTS 100 E. Coli HY transcription andtranslation kits from Roche Diagnostics Ltd., Lewes, UK and the TNTQuick coupled Transcription/Translation System from Promega UK,Southampton, UK).

The expression vector can be produced according to a method known in theart. For example, the vector can be produced by (1) excising a DNAfragment containing a DNA of the present invention or a gene containinga DNA of the present invention and (2) ligating the DNA fragmentdownstream of the promoter in an appropriate expression vector. A widevariety of expression systems can be used, such as and withoutlimitation, chromosomal, episomal and virus-derived systems, e.g.plasmids derived from Escherichia coli (e.g. pBR322, pBR325, pUC18, andpUC118), plasmids derived from Bacillus subtilis (e.g. pUB110, pTP5, andpC194), from bacteriophage, from transposons, from yeast episomes (e.g.pSH19 and pSH15), from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage (such as [lambda]phage) genetic elements, such as cosmids and phagemids. The expressionsystems may contain control regions that regulate as well as engenderexpression. Promoters to be used in the present invention may be anypromoters as long as they are appropriate for hosts to be used for geneexpression. For example, when a host is Escherichia coli, a trppromoter, a lac promoter, a recA promoter, a pL promoter, an 1 pppromoter, and the like are preferred. When a host is Bacillus subtilis,an SPO1 promoter, an SPO2 promoter, a penP promoter, and the like arepreferred. When a host is yeast, a PHOS promoter, a PGK promoter, a GAPpromoter, an ADH promoter, and the like are preferred. When an animalcell is used as a host, examples of promoters for use in this caseinclude an SRa promoter, an SV40 promoter, an LTR promoter, a CMVpromoter, and an HSV-TK promoter. Generally, any system or vector thatis able to maintain, propagate or express a nucleic acid to produce apolypeptide in a host may be used.

The appropriate nucleic acid sequence may be inserted into an expressionsystem by any variety of well known and routine techniques, such asthose set forth in Sambrook et al., supra. Appropriate secretion signalsmay be incorporated into the OGTA001 polypeptide to allow secretion ofthe translated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular environment. These signals may beendogenous to the OGTA001 polypeptide or they may be heterologoussignals. Transformation of the host cells can be carried out accordingto methods known in the art. For example, the following documents can bereferred to: Proc. Natl. Acad. Sci. U.S.A., Vol. 69, 2110 (1972); Gene,Vol. 17, 107 (1982); Molecular & General Genetics, Vol. 168, 111 (1979);Methods in Enzymology, Vol. 194, 182-187 (1991); Proc. Natl. Acad. Sci.U.S.A.), Vol. 75, 1929 (1978); Cell Technology, separate volume 8, NewCell Technology, Experimental Protocol. 263-267 (1995) (issued byShujunsha); and Virology, Vol. 52, 456 (1973). The thus obtainedtransformant transformed with an expression vector containing a DNA ofthe present invention or a gene containing a DNA of the presentinvention can be cultured according to a method known in the art. Forexample, when hosts are bacteria of the genus Escherichia, the bacteriaare generally cultured at approximately 15° C. to 43° C. forapproximately 3 to 24 hours. If necessary, aeration or agitation canalso be added. When hosts are bacteria of the genus Bacillus, thebacteria are generally cultured at approximately 30° C. to 40° C. forapproximately 6 to 24 hours. If necessary, aeration or agitation canalso be added. When transformants whose hosts are yeast are cultured,culture is generally carried out at approximately 20° C. to 35° C. forapproximately 24 to 72 hours using media with pH adjusted to beapproximately 5 to 8. If necessary, aeration or agitation can also beadded. When transformants whose hosts are animal cells are cultured, thecells are generally cultured at approximately 30° C. to 40° C. forapproximately 15 to 60 hours using media with the pH adjusted to beapproximately 6 to 8. If necessary, aeration or agitation can also beadded.

If an OGTA001 polypeptide is to be expressed for use in cell-basedscreening assays, it is preferred that the polypeptide be produced atthe cell surface. In this event, the cells may be harvested prior to usein the screening assay. If the OGTA001 polypeptide is secreted into themedium, the medium can be recovered in order to isolate saidpolypeptide. If produced intracellularly, the cells must first be lysedbefore the OGTA001 polypeptide is recovered.

OGTA001 polypeptide can be recovered and purified from recombinant cellcultures or from other biological sources by well known methodsincluding, ammonium sulphate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, affinity chromatography, hydrophobic interactionchromatography, hydroxylapatite chromatography, molecular sievingchromatography, centrifugation methods, electrophoresis methods andlectin chromatography. In one embodiment, a combination of these methodsis used. In another embodiment, high performance liquid chromatographyis used. In a further embodiment, an antibody which specifically bindsto an OGTA001 polypeptide can be used to deplete a sample comprising anOGTA001 polypeptide of said polypeptide or to purify said polypeptide.

To separate and purify a polypeptide or a protein of the presentinvention from the culture products, for example, after culture,microbial bodies or cells are collected by a known method, they aresuspended in an appropriate buffer, the microbial bodies or the cellsare disrupted by, for example, ultrasonic waves, lysozymes, and/orfreeze-thawing, the resultant is then subjected to centrifugation orfiltration, and then a crude extract of the protein can be obtained. Thebuffer may also contain a protein denaturation agent such as urea orguanidine hydrochloride or a surfactant such as Triton X-100™. When theprotein is secreted in a culture solution, microbial bodies or cells anda supernatant are separated by a known method after the completion ofculture and then the supernatant is collected. The protein contained inthe thus obtained culture supernatant or the extract can be purified byan appropriate combination of known separation and purification methods.The thus obtained polypeptide (protein) of the present invention can beconverted into a salt by a known method or a method according thereto.Conversely, when the polypeptide (protein) of the present invention isobtained in the form of a salt, it can be converted into a free proteinor peptide or another salt by a known method or a method accordingthereto. Moreover, an appropriate protein modification enzyme such astrypsin or chymotrypsin is caused to act on a protein produced by arecombinant before or after purification, so that modification can bearbitrarily added or a polypeptide can be partially removed. Thepresence of a polypeptide (protein) of the present invention or a saltthereof can be measured by various binding assays, enzyme immunoassaysusing specific antibodies, and the like.

Techniques well known in the art may be used for refolding to regeneratenative or active conformations of the OGTA001 polypeptide when thepolypeptide has been denatured during isolation and or purification. Inthe context of the present invention, OGTA001 polypeptide can beobtained from a biological sample from any source, such as and withoutlimitation, a blood sample or tissue sample, e.g. a colorectal tissuesample.

OGTA001 polypeptide may be in the form of a “mature protein” or may bepart of a larger protein such as a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, a pre-, pro- or prepro-protein sequence,or a sequence which aids in purification such as an affinity tag, forexample, but without limitation, multiple histidine residues, a FLAGtag, HA tag or myc tag.

An additional sequence that may provide stability during recombinantproduction may also be used. Such sequences may be optionally removed asrequired by incorporating a cleavable sequence as an additional sequenceor part thereof. Thus, an OGTA001 polypeptide may be fused to othermoieties including other polypeptides or proteins (for example,glutathione S-transferase and protein A). Such a fusion protein can becleaved using an appropriate protease, and then separated into eachprotein. Such additional sequences and affinity tags are well known inthe art. In addition to the above, features known in the art, such as anenhancer, a splicing signal, a polyA addition signal, a selectionmarker, and an SV40 replication origin can be added to an expressionvector, if desired.

Production of Affinity Reagents to OGTA001

According to those in the art, there are three main types of affinityreagent—monoclonal antibodies, phage display antibodies and smallmolecules such as Affibodies, Domain Antibodies (dAbs), Nanobodies orUnibodies. In general in applications according to the present inventionwhere the use of antibodies is stated, other affinity reagents (e.g.Affibodies, domain antibodies, Nanobodies or Unibodies) may be employed.

Production of Antibodies to OGTA001

According to the invention OGTA001, an OGTA001 analog, anOGTA001-related protein or a fragment or derivative of any of theforegoing may be used as an immunogen to generate antibodies whichimmunospecifically bind such an immunogen. Such immunogens can beisolated by any convenient means, including the methods described above.The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3^(rd) Edition, W.E. Paul, ed., Raven Press,N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush(1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”Antibodies of the invention include, but are not limited to polyclonal,monoclonal, bispecific, humanized or chimeric antibodies, single chainantibodies, Fab fragments and F(ab′)₂ fragments, fragments produced by aFab expression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. The immunoglobulinmolecules of the invention can be of any class (e.g., IgG, IgE, IgM, IgDand IgA) or subclass of immunoglobulin molecule.

The term “specifically binds” (or “immunospecifically binds”) is notintended to indicate that an antibody binds exclusively to its intendedtarget. Rather, an antibody “specifically binds” if its affinity for itsintended target is about 5-fold greater when compared to its affinityfor a non-target molecule. Preferably the affinity of the antibody willbe at least about 5 fold, preferably 10 fold, more preferably 25-fold,even more preferably 50-fold, and most preferably 100-fold or more,greater for a target molecule than its affinity for a non-targetmolecule. In preferred embodiments, Specific binding between an antibodyor other binding agent and an antigen means a binding affinity of atleast 10⁶ M⁻¹. Preferred antibodies bind with affinities of at leastabout 10⁷ M⁻¹, and preferably between about 10⁸ M⁻¹ to about 10⁹ M⁻¹,about 10⁹ M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ M⁻¹ about 10¹¹ M⁻¹.

Affinity is calculated as K_(d)=k_(off)/k_(on) (k_(off) is thedissociation rate constant, k_(on) is the association rate constant andK_(d) is the equilibrium constant. Affinity can be determined atequilibrium by measuring the fraction bound (r) of labeled ligand atvarious concentrations (c). The data are graphed using the Scatchardequation: r/c=K(n−r):

where

r=moles of bound ligand/mole of receptor at equilibrium;

c=free ligand concentration at equilibrium;

K=equilibrium association constant; and

n=number of ligand binding sites per receptor molecule

By graphical analysis, r/c is plotted on the Y-axis versus r on theX-axis thus producing a Scatchard plot. The affinity is the negativeslope of the line. k_(off) can be determined by competing bound labeledligand with unlabeled excess ligand (see, e.g., U.S. Pat. No.6,316,409). The affinity of a targeting agent for its target molecule ispreferably at least about 1×10⁻⁶ moles/liter, is more preferably atleast about 1×10⁻⁷ moles/liter, is even more preferably at least about1×10⁻⁸ moles/liter, is yet even more preferably at least about 1×10⁻⁹moles/liter, and is most preferably at least about 1×10⁻¹⁰ moles/liter.Antibody affinity measurement by Scatchard analysis is well known in theart. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelsonand Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

In one embodiment, antibodies that recognize gene products of genesencoding OGTA001 are publicly available. In another embodiment, methodsknown to those skilled in the art are used to produce antibodies thatrecognize OGTA001, an OGTA001 analog, an OGTA001-related polypeptide, ora fragment or derivative of any of the foregoing. One skilled in the artwill recognize that many procedures are available for the production ofantibodies, for example, as described in Antibodies, A LaboratoryManual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988),Cold Spring Harbor, N.Y. One skilled in the art will also appreciatethat binding fragments or Fab fragments which mimic antibodies can alsobe prepared from genetic information by various procedures (AntibodyEngineering: A Practical Approach (Borrebaeck, C., ed.), 1995, OxfordUniversity Press, Oxford; J. Immunol. 149, 3914-3920 (1992)).

In one embodiment of the invention, antibodies to a specific domain ofOGTA001 are produced. In a specific embodiment, hydrophilic fragments ofOGTA001 are used as immunogens for antibody production.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). For example, to select antibodieswhich recognize a specific domain of OGTA001, one may assay generatedhybridomas for a product which binds to an OGTA001 fragment containingsuch domain. For selection of an antibody that specifically binds afirst OGTA001 homolog but which does not specifically bind to (or bindsless avidly to) a second OGTA001 homolog, one can select on the basis ofpositive binding to the first OGTA001 homolog and a lack of binding to(or reduced binding to) the second OGTA001 homolog. Similarly, forselection of an antibody that specifically binds OGTA001 but which doesnot specifically bind to (or binds less avidly to) a different isoformof the same protein (such as a different glycoform having the same corepeptide as OGTA001), one can select on the basis of positive binding toOGTA001 and a lack of binding to (or reduced binding to) the differentisoform (e.g., a different glycoform). Thus, the present inventionprovides an antibody (preferably a monoclonal antibody) that binds withgreater affinity (preferably at least 2-fold, more preferably at least5-fold, still more preferably at least 10-fold greater affinity) toOGTA001 than to a different isoform or isoforms (e.g., glycoforms) ofOGTA001.

Polyclonal antibodies which may be used in the methods of the inventionare heterogeneous populations of antibody molecules derived from thesera of immunized animals. Unfractionated immune serum can also be used.Various procedures known in the art may be used for the production ofpolyclonal antibodies to OGTA001, a fragment of OGTA001, anOGTA001-related polypeptide, or a fragment of an OGTA001-relatedpolypeptide. For example, one way is to purify polypeptides of interestor to synthesize the polypeptides of interest using, e.g., solid phasepeptide synthesis methods well known in the art. See, e.g., Guide toProtein Purification, Murray P. Deutcher, ed., Meth. Enzymol. Vol 182(1990); Solid Phase Peptide Synthesis, Greg B. Fields ed., Meth.Enzymol. Vol 289 (1997); Kiso et al., Chem. Pharm. Bull. (Tokyo) 38:1192-99, 1990; Mostafavi et al., Biomed. Pept. Proteins Nucleic Acids 1:255-60, 1995; Fujiwara et al., Chem. Pharm. Bull. (Tokyo) 44: 1326-31,1996. The selected polypeptides may then be used to immunize byinjection various host animals, including but not limited to rabbits,mice, rats, etc., to generate polyclonal or monoclonal antibodies. ThePreferred Technology described herein in Example 1 provides isolatedOGTA001 suitable for such immunization. If OGTA001 is purified by gelelectrophoresis, OGTA001 can be used for immunization with or withoutprior extraction from the polyacrylamide gel. Various adjuvants (i.e.immunostimulants) may be used to enhance the immunological response,depending on the host species, including, but not limited to, completeor incomplete Freund's adjuvant, a mineral gel such as aluminumhydroxide, surface active substance such as lysolecithin, pluronicpolyol, a polyanion, a peptide, an oil emulsion, keyhole limpethemocyanin, dinitrophenol, and an adjuvant such as BCG (bacilleCalmette-Guerin) or corynebacterium parvum. Additional adjuvants arealso well known in the art.

For preparation of monoclonal antibodies (mAbs) directed toward OGTA001,a fragment of OGTA001, an OGTA001-related polypeptide, or a fragment ofan OGTA001-related polypeptide, any technique which provides for theproduction of antibody molecules by continuous cell lines in culture maybe used. For example, the hybridoma technique originally developed byKohler and Milstein (1975, Nature 256:495-497), as well as the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. The hybridoma producing the mAbs of the invention maybe cultivated in vitro or in vivo. In an additional embodiment of theinvention, monoclonal antibodies can be produced in germ-free animalsutilizing known technology (PCT/US90/02545, incorporated herein byreference).

The monoclonal antibodies include but are not limited to humanmonoclonal antibodies and chimeric monoclonal antibodies (e.g.,human-mouse chimeras). A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a human immunoglobulin constant region and a variableregion derived from a murine mAb. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.)

Chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in PCT Publication No. WO 87/02671; European PatentApplication 184,187; European Patent Application 171,496; EuropeanPatent Application 173,494; PCT Publication No. WO 86/01533; U.S. Pat.No. 4,816,567; European Patent Application 125,023; Better et al., 1988,Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987,Canc. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shawet al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985,Science 229:1202-1207; Oi et al., 1986, Bio/Techniques 4:214; U.S. Pat.No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al., 1988, J. Immunol.141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human subjects. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chain genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion ofOGTA001. Monoclonal antibodies directed against the antigen can beobtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.) andGenpharm (San Jose, Calif.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al. (1994) Bio/technology12:899-903).

The antibodies of the present invention can also be generated by the useof phage display technology to produce and screen libraries ofpolypeptides for binding to a selected target. See, e.g., Cwirla et al.,Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; Devlin et al., Science249, 404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladneret al., U.S. Pat. No. 5,571,698. A basic concept of phage displaymethods is the establishment of a physical association between DNAencoding a polypeptide to be screened and the polypeptide. This physicalassociation is provided by the phage particle, which displays apolypeptide as part of a capsid enclosing the phage genome which encodesthe polypeptide. The establishment of a physical association betweenpolypeptides and their genetic material allows simultaneous massscreening of very large numbers of phage bearing different polypeptides.Phage displaying a polypeptide with affinity to a target bind to thetarget and these phage are enriched by affinity screening to the target.The identity of polypeptides displayed from these phage can bedetermined from their respective genomes. Using these methods apolypeptide identified as having a binding affinity for a desired targetcan then be synthesized in bulk by conventional means. See, e.g., U.S.Pat. No. 6,057,098, which is hereby incorporated in its entirety,including all tables, figures, and claims. In particular, such phage canbe utilized to display antigen binding domains expressed from arepertoire or combinatorial antibody library (e.g., human or murine).Phage expressing an antigen binding domain that binds the antigen ofinterest can be selected or identified with antigen, e.g., using labeledantigen or antigen bound or captured to a solid surface or bead. Phageused in these methods are typically filamentous phage including fd andM13 binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Phage display methods that can be used tomake the antibodies of the present invention include those disclosed inBrinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J.Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J.Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burtonet al., Advances in Immunology 57:191-280 (1994); PCT Application No.PCT/GB91/01134; PCT Publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988).

The invention further provides for the use of bispecific antibodies,which can be made by methods known in the art. Traditional production offull length bispecific antibodies is based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Milstein et al., 1983, Nature 305:537-539).Because of the random assortment of immunoglobulin heavy and lightchains, these hybridomas (quadromas) produce a potential mixture of 10different antibody molecules, of which only one has the correctbispecific structure. Purification of the correct molecule, which isusually done by affinity chromatography steps, is rather cumbersome, andthe product yields are low. Similar procedures are disclosed in WO93/08829, published 13 May 1993, and in Traunecker et al., 1991, EMBO J.10:3655-3659.

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690 published Mar. 3,1994. For further details for generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology, 1986, 121:210.

The invention provides functionally active fragments, derivatives oranalogs of the anti-OGTA001 immunoglobulin molecules. Functionallyactive means that the fragment, derivative or analog is able to elicitanti-anti-idiotype antibodies (i.e., tertiary antibodies) that recognizethe same antigen that is recognized by the antibody from which thefragment, derivative or analog is derived. Specifically, in a preferredembodiment the antigenicity of the idiotype of the immunoglobulinmolecule may be enhanced by deletion of framework and CDR sequences thatare C-terminal to the CDR sequence that specifically recognizes theantigen. To determine which CDR sequences bind the antigen, syntheticpeptides containing the CDR sequences can be used in binding assays withthe antigen by any binding assay method known in the art.

The present invention provides antibody fragments such as, but notlimited to, F(ab′)₂ fragments and Fab fragments. Antibody fragmentswhich recognize specific epitopes may be generated by known techniques.F(ab′)₂ fragments consist of the variable region, the light chainconstant region and the CH1 domain of the heavy chain and are generatedby pepsin digestion of the antibody molecule. Fab fragments aregenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.The invention also provides heavy chain and light chain dimers of theantibodies of the invention, or any minimal fragment thereof such as Fvsor single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature334:544-54), or any other molecule with the same specificity as theantibody of the invention. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may be used (Skerra etal., 1988, Science 242:1038-1041).

In other embodiments, the invention provides fusion proteins of theimmunoglobulins of the invention (or functionally active fragmentsthereof), for example in which the immunoglobulin is fused via acovalent bond (e.g., a peptide bond), at either the N-terminus or theC-terminus to an amino acid sequence of another protein (or portionthereof, preferably at least 10, 20 or 50 amino acid portion of theprotein) that is not the immunoglobulin. Preferably the immunoglobulin,or fragment thereof, is covalently linked to the other protein at theN-terminus of the constant domain. As stated above, such fusion proteinsmay facilitate purification, increase half-life in vivo, and enhance thedelivery of an antigen across an epithelial barrier to the immunesystem.

The immunoglobulins of the invention include analogs and derivativesthat are modified, i.e., by the covalent attachment of any type ofmolecule as long as such covalent attachment does not impairimmunospecific binding. For example, but not by way of limitation, thederivatives and analogs of the immunoglobulins include those that havebeen further modified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, etc. Additionally, the analog orderivative may contain one or more non-classical amino acids.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of OGTA001, e.g., for imagingthis protein, measuring levels thereof in appropriate physiologicalsamples, in diagnostic methods, etc.

Production of Affibodies to OGTA001

Affibody molecules represent a new class of affinity proteins based on a58-amino acid residue protein domain, derived from one of theIgG-binding domains of staphylococcal protein A. This three helix bundledomain has been used as a scaffold for the construction of combinatorialphagemid libraries, from which Affibody variants that target the desiredmolecules can be selected using phage display technology (Nord K,Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren P A, Bindingproteins selected from combinatorial libraries of an α-helical bacterialreceptor domain, Nat Biotechnol 1997; 15:772-7. Ronmark J, Gronlund H,Uhlen M, Nygren P A, Human immunoglobulin A (IgA)-specific ligands fromcombinatorial engineering of protein A, Eur J Biochem 2002;269:2647-55.). The simple, robust structure of Affibody molecules incombination with their low molecular weight (6 kDa), make them suitablefor a wide variety of applications, for instance, as detection reagents(Ronmark J, Hansson M, Nguyen T, et al, Construction andcharacterization of affibody-Fc chimeras produced in Escherichia coli,Immunol Methods 2002; 261:199-211) and to inhibit receptor interactions(Sandstorm K, Xu Z, Forsberg G, Nygren P A, Inhibition of the CD28-CD80co-stimulation signal by a CD28-binding Affibody ligand developed bycombinatorial protein engineering, Protein Eng 2003; 16:691-7). Furtherdetails of Affibodies and methods of production thereof may be obtainedby reference to U.S. Pat. No. 5,831,012 which is herein incorporated byreference in its entirety.

Labelled Affibodies may also be useful in imaging applications fordetermining abundance of Isoforms.

Production of Domain Antibodies to OGTA001

References to antibodies herein embrace references to Domain Antibodies.Domain Antibodies (dAbs) are the smallest functional binding units ofantibodies, corresponding to the variable regions of either the heavy(VH) or light (VL) chains of human antibodies. Domain Antibodies have amolecular weight of approximately 13 kDa. Domantis has developed aseries of large and highly functional libraries of fully human V_(H) andV_(L) dAbs (more than ten billion different sequences in each library),and uses these libraries to select dAbs that are specific to therapeutictargets. In contrast to many conventional antibodies, Domain Antibodiesare well expressed in bacterial, yeast, and mammalian cell systems.Further details of domain antibodies and methods of production thereofmay be obtained by reference to U.S. Pat. No. 6,291,158; U.S. Pat. No.6,582,915; U.S. Pat. No. 6,593,081; U.S. Pat. No. 6,172,197; U.S. Pat.No. 6,696,245; US Serial No. 2004/0110941; European patent applicationNo. 1433846 and European Patents 0368684 & 0616640; WO05/035572,WO04/101790, WO04/081026, WO04/058821, WO04/003019 and WO03/002609, eachof which is herein incorporated by reference in its entirety.

Production of Nanobodies to OGTA001

Nanobodies are antibody-derived therapeutic proteins that contain theunique structural and functional properties of naturally-occurringheavy-chain antibodies. These heavy-chain antibodies contain a singlevariable domain (VHH) and two constant domains (C_(H)2 and C_(H)3).Importantly, the cloned and isolated VIM domain is a perfectly stablepolypeptide harbouring the full antigen-binding capacity of the originalheavy-chain antibody. Nanobodies have a high homology with the VHdomains of human antibodies and can be further humanised without anyloss of activity. Importantly, Nanobodies have a low immunogenicpotential, which has been confirmed in primate studies with Nanobodylead compounds.

Nanobodies combine the advantages of conventional antibodies withimportant features of small molecule drugs. Like conventionalantibodies, Nanobodies show high target specificity, high affinity fortheir target and low inherent toxicity. However, like small moleculedrugs they can inhibit enzymes and readily access receptor clefts.Furthermore, Nanobodies are extremely stable, can be administered bymeans other than injection (see e.g. WO 04/041867, which is hereinincorporated by reference in its entirety) and are easy to manufacture.Other advantages of Nanobodies include recognising uncommon or hiddenepitopes as a result of their small size, binding into cavities oractive sites of protein targets with high affinity and selectivity dueto their unique 3-dimensional, drug format flexibility, tailoring ofhalf-life and ease and speed of drug discovery.

Nanobodies are encoded by single genes and are efficiently produced inalmost all prokaryotic and eukaryotic hosts e.g. E. coli (see e.g. U.S.Pat. No. 6,765,087, which is herein incorporated by reference in itsentirety), moulds (for example Aspergillus or Trichoderma) and yeast(for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (seee.g. U.S. Pat. No. 6,838,254, which is herein incorporated by referencein its entirety). The production process is scalable and multi-kilogramquantities of Nanobodies have been produced. Because Nanobodies exhibita superior stability compared with conventional antibodies, they can beformulated as a long shelf-life, ready-to-use solution.

The Nanoclone method (see e.g. WO 06/079372, which is hereinincorporated bye reference in its entirety) is a proprietary method forgenerating Nanobodies against a desired target, based on automatedhigh-throughout selection of B-cells.

Production of Unibodies to OGTA001

UniBody is a new proprietary antibody technology that creates a stable,smaller antibody format with an anticipated longer therapeutic windowthan current small antibody formats. IgG4 antibodies are consideredinert and thus do not interact with the immune system. Genmab modifiedfully human IgG4 antibodies by eliminating the hinge region of theantibody. Unlike the full size IgG4 antibody, the half molecule fragmentis very stable and is termed a UniBody. Halving the IgG4 molecule leftonly one area on the UniBody that can bind to disease targets and theUniBody therefore binds univalently to only one site on target cells.This univalent binding does not stimulate cancer cells to grow likebivalent antibodies might and opens the door for treatment of some typesof cancer which ordinary antibodies cannot treat.

The UniBody is about half the size of a regular IgG4 antibody. Thissmall size can be a great benefit when treating some forms of cancer,allowing for better distribution of the molecule over larger solidtumors and potentially increasing efficacy.

Fabs typically do not have a very long half-life. UniBodies, however,were cleared at a similar rate to whole IgG4 antibodies and were able tobind as well as whole antibodies and antibody fragments in pre-clinicalstudies. Other antibodies primarily work by killing the targeted cellswhereas UniBodies only inhibit or silence the cells.

Further details of Unibodies may be obtained by reference to patentWO2007/059782, which is herein incorporated by reference in itsentirety.

Expression of Affinity Reagents Expression of Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or by recombinant expression, and are preferably produced byrecombinant expression techniques.

Recombinant expression of antibodies, or fragments, derivatives oranalogs thereof, requires construction of a nucleic acid that encodesthe antibody. If the nucleotide sequence of the antibody is known, anucleic acid encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,1994, BioTechniques 17:242), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding antibody, annealing and ligation of those oligonucleotides, andthen amplification of the ligated oligonucleotides by PCR.

Alternatively, the nucleic acid encoding the antibody may be obtained bycloning the antibody. If a clone containing the nucleic acid encodingthe particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may beobtained from a suitable source (e.g., an antibody cDNA library, or cDNAlibrary generated from any tissue or cells expressing the antibody) byPCR amplification using synthetic primers hybridizable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence.

If an antibody molecule that specifically recognizes a particularantigen is not available (or a source for a cDNA library for cloning anucleic acid encoding such an antibody), antibodies specific for aparticular antigen may be generated by any method known in the art, forexample, by immunizing an animal, such as a rabbit, to generatepolyclonal antibodies or, more preferably, by generating monoclonalantibodies. Alternatively, a clone encoding at least the Fab portion ofthe antibody may be obtained by screening Fab expression libraries(e.g., as described in Huse et al., 1989, Science 246:1275-1281) forclones of Fab fragments that bind the specific antigen or by screeningantibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624;Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid encoding at least the variable domain of theantibody molecule is obtained, it may be introduced into a vectorcontaining the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g., PCT Publication WO 86/05807; PCTPublication WO 89/01036; and U.S. Pat. No. 5,122,464). Vectorscontaining the complete light or heavy chain for co-expression with thenucleic acid to allow the expression of a complete antibody molecule arealso available. Then, the nucleic acid encoding the antibody can be usedto introduce the nucleotide substitution(s) or deletion(s) necessary tosubstitute (or delete) the one or more variable region cysteine residuesparticipating in an intrachain disulfide bond with an amino acid residuethat does not contain a sulfhydyl group. Such modifications can becarried out by any method known in the art for the introduction ofspecific mutations or deletions in a nucleotide sequence, for example,but not limited to, chemical mutagenesis, in vitro site directedmutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551), PCTbased methods, etc.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human antibodyconstant region, e.g., humanized antibodies.

Once a nucleic acid encoding an antibody molecule of the invention hasbeen obtained, the vector for the production of the antibody moleculemay be produced by recombinant DNA technology using techniques wellknown in the art. Thus, methods for preparing the protein of theinvention by expressing nucleic acid containing the antibody moleculesequences are described herein. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining an antibody molecule coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al. (1990, Molecular Cloning, ALaboratory Manual, 2^(nd) Ed., Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.) and Ausubel et al. (eds., 1998, Current Protocolsin Molecular Biology, John Wiley & Sons, NY).

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention.

The host cells used to express a recombinant antibody of the inventionmay be either bacterial cells such as Escherichia coli, or, preferably,eukaryotic cells, especially for the expression of whole recombinantantibody molecule. In particular, mammalian cells such as Chinesehamster ovary cells (CHO), in conjunction with a vector such as themajor intermediate early gene promoter element from humancytomegalovirus are an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

A variety of host-expression vector systems may be utilized to expressan antibody molecule of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express the antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions comprising an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). In mammalian host cells, a number ofviral-based expression systems (e.g., an adenovirus expression system)may be utilized.

As discussed above, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein.

For long-term, high-yield production of recombinant antibodies, stableexpression is preferred. For example, cell lines that stably express anantibody of interest can be produced by transfecting the cells with anexpression vector comprising the nucleotide sequence of the antibody andthe nucleotide sequence of a selectable (e.g., neomycin or hygromycin),and selecting for expression of the selectable marker. Such engineeredcell lines may be particularly useful in screening and evaluation ofcompounds that interact directly or indirectly with the antibodymolecule.

The expression levels of the antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once the antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an antibody molecule, for example, by chromatography(e.g., ion exchange chromatography, affinity chromatography such as withprotein A or specific antigen, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins.

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897).In this system, the gene of interest is subcloned into a vacciniarecombination plasmid such that the open reading frame of the gene istranslationally fused to an amino-terminal tag consisting of sixhistidine residues. The tag serves as a matrix binding domain for thefusion protein. Extracts from cells infected with recombinant vacciniavirus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns andhistidine-tagged proteins are selectively eluted withimidazole-containing buffers.

The antibodies that are generated by these methods may then be selectedby first screening for affinity and specificity with the purifiedpolypeptide of interest and, if required, comparing the results to theaffinity and specificity of the antibodies with polypeptides that aredesired to be excluded from binding. The screening procedure can involveimmobilization of the purified polypeptides in separate wells ofmicrotiter plates. The solution containing a potential antibody orgroups of antibodies is then placed into the respective microtiter wellsand incubated for about 30 min to 2 h. The microtiter wells are thenwashed and a labeled secondary antibody (for example, an anti-mouseantibody conjugated to alkaline phosphatase if the raised antibodies aremouse antibodies) is added to the wells and incubated for about 30 minand then washed. Substrate is added to the wells and a color reactionwill appear where antibody to the immobilized polypeptide(s) is present.

The antibodies so identified may then be further analyzed for affinityand specificity in the assay design selected. In the development ofimmunoassays for a target protein, the purified target protein acts as astandard with which to judge the sensitivity and specificity of theimmunoassay using the antibodies that have been selected. Because thebinding affinity of various antibodies may differ; certain antibodypairs (e.g., in sandwich assays) may interfere with one anothersterically, etc., assay performance of an antibody may be a moreimportant measure than absolute affinity and specificity of an antibody.

Those skilled in the art will recognize that many approaches can betaken in producing antibodies or binding fragments and screening andselecting for affinity and specificity for the various polypeptides, butthese approaches do not change the scope of the invention.

For therapeutic applications, antibodies (particularly monoclonalantibodies) may suitably be human or humanized animal (e.g. mouse)antibodies. Animal antibodies may be raised in animals using the humanprotein (e.g. OGTA001) as immunogen. Humanisation typically involvesgrafting CDRs identified thereby into human framework regions. Normallysome subsequent retromutation to optimize the conformation of chains isrequired. Such processes are known to persons skilled in the art.

Expression of Affibodies

The construction of affibodies has been described elsewhere (Ronnmark J,Gronlund H, Uhle'n, M., Nygren P. A°, Human immunoglobulin A(IgA)-specific ligands from combinatorial engineering of protein A,2002, Eur. J. Biochem. 269, 2647-2655.), including the construction ofaffibody phage display libraries (Nord, K., Nilsson, J., Nilsson, B.,Uhle'n, M. & Nygren, P. A°, A combinatorial library of an a-helicalbacterial receptor domain, 1995, Protein Eng. 8, 601-608. Nord, K.,Gunneriusson, E., Ringdahl, J., Sta°hl, S., Uhle'n, M. & Nygren, P. A°,Binding proteins selected from combinatorial libraries of an a-helicalbacterial receptor domain, 1997, Nat. Biotechnol. 15, 772-777.)

The biosensor analyses to investigate the optimal affibody variantsusing biosensor binding studies has also been described elsewhere(Ronnmark J, Gronlund H, Uhle'n, M., Nygren P. A°, Human immunoglobulinA (IgA)-specific ligands from combinatorial engineering of protein A,2002, Eur. J. Biochem. 269, 2647-2655.).

Conjugated Affinity Reagents

In a preferred embodiment, anti-OGTA001 affinity reagents such asantibodies or fragments thereof are conjugated to a diagnostic ortherapeutic moiety. The antibodies can be used for diagnosis or todetermine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive nuclides, positron emitting metals (for use in positronemission tomography), and nonradioactive paramagnetic metal ions. Seegenerally U.S. Pat. No. 4,741,900 for metal ions which can be conjugatedto antibodies for use as diagnostics according to the present invention.Suitable enzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; suitable prosthetic groupsinclude streptavidin, avidin and biotin; suitable fluorescent materialsinclude umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride andphycoerythrin; suitable luminescent materials include luminol; suitablebioluminescent materials include luciferase, luciferin, and aequorin;and suitable radioactive nuclides include ¹²⁵I, ¹³¹I, ¹¹¹In and ⁹⁹Tc.⁶⁸Ga may also be employed.

Anti-OGTA001 antibodies or fragments thereof can be conjugated to atherapeutic agent or drug moiety to modify a given biological response.The therapeutic agent or drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, -interferon, -interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator, athrombotic agent or an anti-angiogenic agent, e.g., angiostatin orendostatin; or, a biological response modifier such as a lymphokine,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), nerve growth factor (NGF) or othergrowth factor.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2^(nd) Ed.), Robinson et al. (eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

An antibody with or without a therapeutic moiety conjugated to it can beused as a therapeutic that is administered alone or in combination withcytotoxic factor(s) and/or cytokine(s).

Diagnosis of Colorectal Cancer

In accordance with the present invention, test samples of colorectaltissue, serum, plasma or urine obtained from a subject suspected ofhaving or known to have colorectal cancer can be used for diagnosis ormonitoring. In one embodiment, a change in the abundance of OGTA001 in atest sample relative to a control sample (from a subject or subjectsfree from colorectal cancer) or a previously determined reference rangeindicates the presence of colorectal cancer. In another embodiment, therelative abundance of OGTA001 in a test sample compared to a controlsample or a previously determined reference range indicates a subtype ofcolorectal cancer (e.g., familial or sporadic colorectal cancer). In yetanother embodiment, the relative abundance of OGTA001 in a test samplerelative to a control sample or a previously determined reference rangeindicates the degree or severity of colorectal cancer (e.g., thelikelihood for metastasis). In any of the aforesaid methods, detectionof OGTA001 may optionally be combined with detection of one or more ofadditional biomarkers for colorectal cancer. Any suitable method in theart can be employed to measure the level of OGTA001, including but notlimited to the Preferred Technologies described herein, kinase assays,immunoassays to detect and/or visualize the OGTA001 (e.g., Western blot,immunoprecipitation followed by sodium dodecyl sulfate polyacrylamidegel electrophoresis, immunocytochemistry, etc.). In a furtherembodiment, a change in the abundance of mRNA encoding OGTA001 in a testsample relative to a control sample or a previously determined referencerange indicates the presence of colorectal cancer. Any suitablehybridization assay can be used to detect OGTA001 expression bydetecting and/or visualizing mRNA encoding the OGTA001 (e.g., Northernassays, dot blots, in situ hybridization, etc.).

In another embodiment of the invention, labeled antibodies (or otheraffinity reagents such as Affibodies, Nanobodies or Unibodies),derivatives and analogs thereof, which specifically bind to OGTA001 canbe used for diagnostic purposes to detect, diagnose, or monitorcolorectal cancer. Preferably, colorectal cancer is detected in ananimal, more preferably in a mammal and most preferably in a human.

Screening Assays

The invention provides methods for identifying agents (e.g., candidatecompounds or test compounds) that bind to OGTA001 or have a stimulatoryor inhibitory effect on the expression or activity of OGTA001. Theinvention also provides methods of identifying agents, candidatecompounds or test compounds that bind to an OGTA001-related polypeptideor an OGTA001 fusion protein or have a stimulatory or inhibitory effecton the expression or activity of an OGTA001-related polypeptide or anOGTA001 fusion protein. Examples of agents, candidate compounds or testcompounds include, but are not limited to, nucleic acids (e.g., DNA andRNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, smallmolecules and other drugs. Agents can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145;U.S. Pat. No. 5,738,996; and U.S. Pat. No. 5,807,683, each of which isincorporated herein in its entirety by reference).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad.Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993,Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl.33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al., 1994, J. Med. Chem. 37:1233, each of which isincorporated herein in its entirety by reference.

Libraries of compounds may be presented, e.g., presented in solution(e.g., Houghten, 1992, Bio/Techniques 13:412-421), or on beads (Lam,1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698;5,403,484; and 5,223,409), plasmids (Cull et al., 1992, Proc. Natl.Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith, 1990, Science249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990,Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J. Mol. Biol.222:301-310), each of which is incorporated herein in its entirety byreference.

In one embodiment, agents that interact with (i.e., bind to) OGTA001, anOGTA001 fragment (e.g. a functionally active fragment), anOGTA001-related polypeptide, a fragment of an OGTA001-relatedpolypeptide, or an OGTA001 fusion protein are identified in a cell-basedassay system. In accordance with this embodiment, cells expressingOGTA001, a fragment of an OGTA001, an OGTA001-related polypeptide, afragment of the OGTA001-related polypeptide, or an OGTA001 fusionprotein are contacted with a candidate compound or a control compoundand the ability of the candidate compound to interact with OGTA001 isdetermined. If desired, this assay may be used to screen a plurality(e.g. a library) of candidate compounds. The cell, for example, can beof prokaryotic origin (e.g., E. coli) or eukaryotic origin (e.g., yeastor mammalian). Further, the cells can express OGTA001, a fragment ofOGTA001, an OGTA001-related polypeptide, a fragment of theOGTA001-related polypeptide, or an OGTA001 fusion protein endogenouslyor be genetically engineered to express OGTA001, a fragment of OGTA001,an OGTA001-related polypeptide, a fragment of the OGTA001-relatedpolypeptide, or an OGTA001 fusion protein. In certain instances,OGTA001, a fragment of the OGTA001, an OGTA001-related polypeptide, afragment of the OGTA001-related polypeptide, or an OGTA001 fusionprotein or the candidate compound is labeled, for example with aradioactive label (such as ³²P, ³⁵S, and ¹²⁵I) or a fluorescent label(such as fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to enabledetection of an interaction between OGTA001 and a candidate compound.The ability of the candidate compound to interact directly or indirectlywith OGTA001, a fragment of OGTA001, an OGTA001-related polypeptide, afragment of an OGTA001-related polypeptide, or an OGTA001 fusion proteincan be determined by methods known to those of skill in the art. Forexample, the interaction between a candidate compound and OGTA001, anOGTA001-related polypeptide, a fragment of an OGTA001-relatedpolypeptide, or an OGTA001 fusion protein can be determined by flowcytometry, a scintillation assay, immunoprecipitation or western blotanalysis.

In another embodiment, agents that interact with (i.e., bind to)OGTA001, an OGTA001 fragment (e.g., a functionally active fragment), anOGTA001-related polypeptide, a fragment of an OGTA001-relatedpolypeptide, or an OGTA001 fusion protein are identified in a cell-freeassay system. In accordance with this embodiment, a native orrecombinant OGTA001 or fragment thereof, or a native or recombinantOGTA001-related polypeptide or fragment thereof, or an OGTA001-fusionprotein or fragment thereof, is contacted with a candidate compound or acontrol compound and the ability of the candidate compound to interactwith OGTA001 or OGTA001-related polypeptide, or OGTA001 fusion proteinis determined. If desired, this assay may be used to screen a plurality(e.g. a library) of candidate compounds. Preferably, OGTA001, an OGTA001fragment, an OGTA001-related polypeptide, a fragment of anOGTA001-related polypeptide, or an OGTA001-fusion protein is firstimmobilized, by, for example, contacting OGTA001, an OGTA001 fragment,an OGTA001-related polypeptide, a fragment of an OGTA001-relatedpolypeptide, or an OGTA001 fusion protein with an immobilized antibody(or other affinity reagent such as an Affibody, Nanobody or Unibody)which specifically recognizes and binds it, or by contacting a purifiedpreparation of OGTA001, an OGTA001 fragment, an OGTA001-relatedpolypeptide, a fragment of an OGTA001-related polypeptide, or an OGTA001fusion protein with a surface designed to bind proteins. OGTA001, anOGTA001 fragment, an OGTA001-related polypeptide, a fragment of anOGTA001-related polypeptide, or an OGTA001 fusion protein may bepartially or completely purified (e.g., partially or completely free ofother polypeptides) or part of a cell lysate. Further, OGTA001, anOGTA001 fragment, an OGTA001-related polypeptide, a fragment of anOGTA001-related polypeptide may be a fusion protein comprising OGTA001or a biologically active portion thereof, or OGTA001-related polypeptideand a domain such as glutathionine-5-transferase. Alternatively,OGTA001, an OGTA001 fragment, an OGTA001-related polypeptide, a fragmentof an OGTA001-related polypeptide or an OGTA001 fusion protein can bebiotinylated using techniques well known to those of skill in the art(e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.). The abilityof the candidate compound to interact with OGTA001, an OGTA001 fragment,an OGTA001-related polypeptide, a fragment of an OGTA001-relatedpolypeptide, or an OGTA001 fusion protein can be determined by methodsknown to those of skill in the art.

In another embodiment, a cell-based assay system is used to identifyagents that bind to or modulate the activity of a protein, such as anenzyme, or a biologically active portion thereof, which is responsiblefor the production or degradation of OGTA001 or is responsible for thepost-translational modification of OGTA001. In a primary screen, aplurality (e.g., a library) of compounds are contacted with cells thatnaturally or recombinantly express: (i) OGTA001, an isoform of OGTA001,an OGTA001 homolog, an OGTA001-related polypeptide, an OGTA001 fusionprotein, or a biologically active fragment of any of the foregoing; and(ii) a protein that is responsible for processing of OGTA001, theOGTA001 isoform, the OGTA001 homolog, the OGTA001-related polypeptide,the OGTA001 fusion protein, or fragment in order to identify compoundsthat modulate the production, degradation, or post-translationalmodification of OGTA001, the OGTA001 isoform, the OGTA001 homolog, theOGTA001-related polypeptide, the OGTA001 fusion protein or fragment. Ifdesired, compounds identified in the primary screen can then be assayedin a secondary screen against cells naturally or recombinantlyexpressing OGTA001. The ability of the candidate compound to modulatethe production, degradation or post-translational modification ofOGTA001, isoform, homolog, OGTA001-related polypeptide, or OGTA001fusion protein can be determined by methods known to those of skill inthe art, including without limitation, flow cytometry, a scintillationassay, immunoprecipitation and western blot analysis.

In another embodiment, agents that competitively interact with (i.e.,bind to) OGTA001, an OGTA001 fragment, an OGTA001-related polypeptide, afragment of an OGTA001-related polypeptide, or an OGTA001 fusion proteinare identified in a competitive binding assay. In accordance with thisembodiment, cells expressing OGTA001, an OGTA001 fragment, anOGTA001-related polypeptide, a fragment of an OGTA001-relatedpolypeptide, or an OGTA001 fusion protein are contacted with a candidatecompound and a compound known to interact with OGTA001, an OGTA001fragment, an OGTA001-related polypeptide, a fragment of anOGTA001-related polypeptide or an OGTA001 fusion protein; the ability ofthe candidate compound to preferentially interact with OGTA001, theOGTA001 fragment, the OGTA001-related polypeptide, the fragment of theOGTA001-related polypeptide, or the OGTA001 fusion protein is thendetermined. Alternatively, agents that preferentially interact with(i.e., bind to) OGTA001, an OGTA001 fragment, an OGTA001-relatedpolypeptide or fragment of an OGTA001-related polypeptide are identifiedin a cell-free assay system by contacting OGTA001, an OGTA001 fragment,an OGTA001-related polypeptide, a fragment of an OGTA001-relatedpolypeptide, or an OGTA001 fusion protein with a candidate compound anda compound known to interact with OGTA001, the OGTA001-relatedpolypeptide or the OGTA001 fusion protein. As stated above, the abilityof the candidate compound to interact with OGTA001, an OGTA001 fragment,an OGTA001-related polypeptide, a fragment of an OGTA001-relatedpolypeptide, or an OGTA001 fusion protein can be determined by methodsknown to those of skill in the art. These assays, whether cell-based orcell-free, can be used to screen a plurality (e.g., a library) ofcandidate compounds.

In another embodiment, agents that modulate (i.e., upregulate ordownregulate) the expression or activity of OGTA001, or anOGTA001-related polypeptide are identified by contacting cells (e.g.,cells of prokaryotic origin or eukaryotic origin) expressing OGTA001, orthe OGTA001-related polypeptide with a candidate compound or a controlcompound (e.g., phosphate buffered saline (PBS)) and determining theexpression of OGTA001, the OGTA001-related polypeptide, or the OGTA001fusion protein, mRNA encoding OGTA001, or mRNA encoding theOGTA001-related polypeptide. The level of expression of OGTA001, theOGTA001-related polypeptide, mRNA encoding OGTA001, or mRNA encoding theOGTA001-related polypeptide in the presence of the candidate compound iscompared to the level of expression of OGTA001, the OGTA001-relatedpolypeptide, mRNA encoding OGTA001, or mRNA encoding the OGTA001-relatedpolypeptide in the absence of the candidate compound (e.g., in thepresence of a control compound). The candidate compound can then beidentified as a modulator of the expression of OGTA001, or theOGTA001-related polypeptide based on this comparison. For example, whenexpression of OGTA001 or mRNA is significantly greater in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of expression of OGTA001 or mRNA.Alternatively, when expression of OGTA001 or mRNA is significantly lessin the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of the expression ofOGTA001 or mRNA. The level of expression of OGTA001 or the mRNA thatencodes it can be determined by methods known to those of skill in theart. For example, mRNA expression can be assessed by Northern blotanalysis or RT-PCR, and protein levels can be assessed by western blotanalysis.

In another embodiment, agents that modulate the activity of OGTA001 oran OGTA001-related polypeptide are identified by contacting apreparation containing OGTA001 or the OGTA001-related polypeptide orcells (e.g., prokaryotic or eukaryotic cells) expressing OGTA001 or theOGTA001-related polypeptide with a test compound or a control compoundand determining the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of OGTA001 or the OGTA001-relatedpolypeptide. The activity of OGTA001 or an OGTA001-related polypeptidecan be assessed by detecting induction of a cellular signal transductionpathway of OGTA001 or the OGTA001-related polypeptide (e.g.,intracellular Ca²⁺, diacylglycerol, IP3, etc.), detecting catalytic orenzymatic activity of the target on a suitable substrate, detecting theinduction of a reporter gene (e.g., a regulatory element that isresponsive to OGTA001 or an OGTA001-related polypeptide and is operablylinked to a nucleic acid encoding a detectable marker, e.g.,luciferase), or detecting a cellular response, for example, cellulardifferentiation, or cell proliferation. Based on the presentdescription, techniques known to those of skill in the art can be usedfor measuring these activities (see, e.g., U.S. Pat. No. 5,401,639,which is incorporated herein by reference). The candidate compound canthen be identified as a modulator of the activity of OGTA001 or anOGTA001-related polypeptide by comparing the effects of the candidatecompound to the control compound. Suitable control compounds includephosphate buffered saline (PBS) and normal saline (NS).

In another embodiment, agents that modulate (i.e., upregulate ordownregulate) the expression, activity or both the expression andactivity of OGTA001 or an OGTA001-related polypeptide are identified inan animal model. Examples of suitable animals include, but are notlimited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and cats.Preferably, the animal used represent a model of colorectal cancer(e.g., xenografts of human colorectal cancer cell lines such asMDA-MB-345 in oestrogen-deprived Severe Combined Immunodeficient (SCID)mice, Eccles et al. 1994 Cell Biophysics 24/25, 279). These can beutilized to test compounds that modulate OGTA001 levels, since thepathology exhibited in these models is similar to that of colorectalcancer. In accordance with this embodiment, the test compound or acontrol compound is administered (e.g., orally, rectally or parenterallysuch as intraperitoneally or intravenously) to a suitable animal and theeffect on the expression, activity or both expression and activity ofOGTA001 or an OGTA001-related polypeptide is determined. Changes in theexpression of OGTA001 or an OGTA001-related polypeptide can be assessedby the methods outlined above.

In yet another embodiment, OGTA001 or an OGTA001-related polypeptide isused as a “bait protein” in a two-hybrid assay or three hybrid assay toidentify other proteins that bind to or interact with OGTA001 or anOGTA001-related polypeptide (see, e.g., U.S. Pat. No. 5,283,317; Zervoset al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924;Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication No. WO94/10300). As those skilled in the art will appreciate, such bindingproteins are also likely to be involved in the propagation of signals byOGTA001 as, for example, upstream or downstream elements of a signalingpathway involving OGTA001.

This invention further provides novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein. In addition, the invention also provides the use of anagent which interacts with, or modulates the activity of, OGTA001 in themanufacture of a medicament for the treatment of colorectal cancer.

Therapeutic Use of OGTA001

The invention provides for treatment or prevention of various diseasesand disorders by administration of a therapeutic compound. Suchcompounds include but are not limited to: OGTA001, OGTA001 analogs,OGTA001-related polypeptides and derivatives (including fragments)thereof; antibodies (or other affinity reagents such as Affibodies,Nanobodies or Unibodies) to the foregoing; nucleic acids encodingOGTA001, OGTA001 analogs, OGTA001-related polypeptides and fragmentsthereof; antisense nucleic acids to a gene encoding OGTA001 or anOGTA001-related polypeptide; and modulator (e.g., agonists andantagonists) of a gene encoding OGTA001 or an OGTA001-relatedpolypeptide. An important feature of the present invention is theidentification of genes encoding OGTA001 involved in colorectal cancer.Colorectal cancer can be treated (e.g. to ameliorate symptoms or toretard onset or progression) or prevented by administration of atherapeutic compound that reduces function or expression of OGTA001 inthe serum or tissue of subjects having colorectal cancer.

In one embodiment, one or more antibodies (or other affinity reagentssuch as Affibodies, Nanobodies or Unibodies) each specifically bindingto OGTA001 are administered alone or in combination with one or moreadditional therapeutic compounds or treatments.

Preferably, a biological product such as an antibody (or other affinityreagent such as an Affibody, Nanobody or Unibody) is allogeneic to thesubject to which it is administered. In a preferred embodiment, a humanOGTA001 or a human OGTA001-related polypeptide, a nucleotide sequenceencoding a human OGTA001 or a human OGTA001-related polypeptide, or anantibody (or other affinity reagent such as an Affibody, Nanobody orUnibody) to a human OGTA001 or a human OGTA001-related polypeptide, isadministered to a human subject for therapy (e.g. to ameliorate symptomsor to retard onset or progression) or prophylaxis.

Without being limited by theory, it is conceived that the therapeuticactivity of antibodies (or other affinity reagents such as Affibodies,Nanobodies or Unibodies) which specifically bind to OGTA001 may beachieved through the phenomenon of Antibody—Dependent Cell-mediatedCytotoxicity (ADCC) (see e.g. Janeway Jr. C. A. et al., Immunobiology,5th ed., 2001, Garland Publishing, ISBN 0-8153-3642-X; Pier G. B. etal., Immunology, Infection, and Immunity, 2004, p246-5; Albanell J. etal., Advances in Experimental Medicine and Biology, 2003, 532:p2153-68and Weng, W.-K. et al., Journal of Clinical Oncology, 2003, 21:p3940-3947).

Treatment and Prevention of Colorectal Cancer

Colorectal cancer is treated or prevented by administration to a subjectsuspected of having or known to have colorectal cancer or to be at riskof developing colorectal cancer of a compound that modulates (i.e.,increases or decreases) the level or activity (i.e., function) ofOGTA001 that is differentially present in the serum or tissue ofsubjects having colorectal cancer compared with serum or tissue ofsubjects free from colorectal cancer. In one embodiment, colorectalcancer is treated or prevented by administering to a subject suspectedof having or known to have colorectal cancer or to be at risk ofdeveloping colorectal cancer a compound that upregulates (i.e.,increases) the level or activity (i.e., function) of OGTA001 that aredecreased in the serum or tissue of subjects having colorectal cancer.Examples of such a compound include, but are not limited to, OGTA001antisense oligonucleotides, ribozymes, antibodies (or other affinityreagents such as Affibodies, Nanobodies or Unibodies) directed againstOGTA001, and compounds that inhibit the enzymatic activity of OGTA001.Other useful compounds e.g., OGTA001 antagonists and small moleculeOGTA001 antagonists, can be identified using in vitro assays.

Colorectal cancer is also treated or prevented by administration to asubject suspected of having or known to have colorectal cancer or to beat risk of developing colorectal cancer of a compound that downregulatesthe level or activity (i.e. function) of OGTA001 that are increased inthe serum or tissue of subjects having colorectal cancer. Examples ofsuch a compound include but are not limited to: OGTA001, OGTA001fragments and OGTA001-related polypeptides; nucleic acids encodingOGTA001, an OGTA001 fragment and an OGTA001-related polypeptide (e.g.,for use in gene therapy); and, for those OGTA001 or OGTA001-relatedpolypeptides with enzymatic activity, compounds or molecules known tomodulate that enzymatic activity. Other compounds that can be used,e.g., OGTA001 agonists, can be identified using in in vitro assays.

In a preferred embodiment, therapy or prophylaxis is tailored to theneeds of an individual subject. Thus, in specific embodiments, compoundsthat promote the level or function of OGTA001 are therapeutically orprophylactically administered to a subject suspected of having or knownto have colorectal cancer, in whom the levels or functions of OGTA001are absent or are decreased relative to a control or normal referencerange. In further embodiments, compounds that promote the level orfunction of OGTA001 are therapeutically or prophylactically administeredto a subject suspected of having or known to have colorectal cancer inwhom the levels or functions of OGTA001 are increased relative to acontrol or to a reference range. In further embodiments, compounds thatdecrease the level or function of OGTA001 are therapeutically orprophylactically administered to a subject suspected of having or knownto have colorectal cancer in whom the levels or functions of OGTA001 areincreased relative to a control or to a reference range. In furtherembodiments, compounds that decrease the level or function of OGTA001are therapeutically or prophylactically administered to a subjectsuspected of having or known to have colorectal cancer in whom thelevels or functions of OGTA001 are decreased relative to a control or toa reference range. The change in OGTA001 function or level due to theadministration of such compounds can be readily detected, e.g., byobtaining a sample (e.g., blood or urine) and assaying in vitro thelevels or activities of OGTA001, or the levels of mRNAs encodingOGTA001, or any combination of the foregoing. Such assays can beperformed before and after the administration of the compound asdescribed herein.

The compounds of the invention include but are not limited to anycompound, e.g., a small organic molecule, protein, peptide, antibody (orother affinity reagent such as an Affibody, Nanobody or Unibody),nucleic acid, etc. that restores the OGTA001 profile towards normal. Thecompounds of the invention may be given in combination with any otherchemotherapy drugs.

Vaccine Therapy

OGTA001 may be useful as antigenic material, and may be used in theproduction of vaccines for treatment or prophylaxis of colorectalcancer. Such material can be “antigenic” and/or “immunogenic”.Generally, “antigenic” is taken to mean that the protein is capable ofbeing used to raise antibodies (or other affinity reagents such asAffibodies, Nanobodies or Unibodies) or indeed is capable of inducing anantibody response in a subject or experimental animal. “Immunogenic” istaken to mean that the protein is capable of eliciting a protectiveimmune response in a subject or experimental animal. Thus, in the lattercase, the protein may be capable of not only generating an antibodyresponse but, in addition, non-antibody based immune responses.“Immunogenic” also embraces whether the protein may elicit animmune-like response in an in-vitro setting eg a T-cell proliferationassay.

The skilled person will appreciate that homologues or derivatives ofOGTA001 will also find use as antigenic/immunogenic material. Thus, forinstance proteins which include one or more additions, deletions,substitutions or the like are encompassed by the present invention. Inaddition, it may be possible to replace one amino acid with another ofsimilar “type”. For instance, replacing one hydrophobic amino acid withanother. One can use a program such as the CLUSTAL program to compareamino acid sequences. This program compares amino acid sequences andfinds the optimal alignment by inserting spaces in either sequence asappropriate. It is possible to calculate amino acid identity orsimilarity (identity plus conservation of amino acid type) for anoptimal alignment. A program like BLASTx will align the longest stretchof similar sequences and assign a value to the fit. It is thus possibleto obtain a comparison where several regions of similarity are found,each having a different score. Both types of analysis are contemplatedin the present invention.

In the case of homologues and derivatives, the degree of identity with aprotein as described herein is less important than that the homologue orderivative should retain its antigenicity and/or immunogenicity.However, suitably, homologues or derivatives having at least 60%similarity (as discussed above) with the proteins or polypeptidesdescribed herein are provided. Preferably, homologues or derivativeshaving at least 70% similarity, more preferably at least 80% similarity,are provided. Most preferably, homologues or derivatives having at least90% or even 95% similarity are provided.

In an alternative approach, the homologues or derivatives could befusion proteins, incorporating moieties which render purificationeasier, for example by effectively tagging the desired protein orpolypeptide. It may be necessary to remove the “tag” or it may be thecase that the fusion protein itself retains sufficient antigenicity tobe useful.

It is well known that it is possible to screen an antigenic protein orpolypeptide to identify epitopic regions, i.e. those regions which areresponsible for the protein or polypeptide's antigenicity orimmunogenicity. Methods well known to the skilled person can be used totest fragments and/or homologues and/or derivatives for antigenicity.Thus, the fragments of the present invention should include one or moresuch epitopic regions or be sufficiently similar to such regions toretain their antigenic/immunogenic properties. Thus, for fragmentsaccording to the present invention the degree of identity is perhapsirrelevant, since they may be 100% identical to a particular part of aprotein or polypeptide, homologue or derivative as described herein. Thekey issue, once again, is that the fragment retains theantigenic/immunogenic properties of the protein from which it isderived.

What is important for homologues, derivatives and fragments is that theypossess at least a degree of the antigenicity/immunogenicity of theprotein or polypeptide from which they are derived. Thus, in anadditional aspect of the invention, there is provided antigenic/orimmunogenic fragments of OGTA001, or of homologues or derivativesthereof.

OGTA001, or antigenic fragments thereof, can be provided alone, as apurified or isolated preparation. They may be provided as part of amixture with one or more other proteins or antigenic fragments thereof.In a further aspect, therefore, the invention provides an antigencomposition comprising OGTA001 and/or one or more antigenic fragmentsthereof. Such a composition can be used for the detection and/ordiagnosis of colorectal cancer.

In a sixth aspect, the present invention provides a method of detectingand/or diagnosing colorectal cancer which comprises:

bringing into contact with a sample to be tested an antigenic OGTA001,or an antigenic fragment thereof, or an antigen composition of theinvention; and

detecting the presence of antibodies (or other affinity reagents such asAffibodies, Nanobodies or Unibodies) to colorectal cancer.

In particular, the protein, antigenic fragment thereof or antigencomposition of the present invention can be used to detect IgA, IgM orIgG antibodies. Suitably, the sample to be tested will be a biologicalsample, e.g. a sample of blood or saliva.

In a further aspect, the invention provides the use of an antigenicOGTA001, antigenic fragment thereof or an antigenic composition of thepresent invention in detecting and/or diagnosing colorectal cancer.Preferably, the detecting and/or diagnosing are carried out in vitro.

The antigenic OGTA001, antigenic fragments thereof or antigeniccomposition of the present invention can be provided as a kit for use inthe in vitro detection and/or diagnosis of colorectal cancer. Thus, in astill further aspect, the present invention provides a kit for use inthe detection and/or diagnosis of colorectal cancer, which kit comprisesan antigenic OGTA001, an antigenic fragment thereof or an antigeniccomposition of the present invention.

In addition, the antigenic OGTA001, antigenic fragment thereof orantigen composition of the invention can be used to induce an immuneresponse against colorectal cancer. Thus, in a yet further aspect, theinvention provides the use of an antigenic OGTA001, an antigenicfragment thereof or an antigen composition of the invention in medicine.

In a further aspect, the present invention provides a compositioncapable of eliciting an immune response in a subject, which compositioncomprises OGTA001, an antigenic fragment thereof, or an antigencomposition of the invention. Suitably, the composition will be avaccine composition, optionally comprising one or more suitableadjuvants. Such a vaccine composition may be either a prophylactic ortherapeutic vaccine composition.

The vaccine compositions of the invention can include one or moreadjuvants. Examples well-known in the art include inorganic gels, suchas aluminium hydroxide, and water-in-oil emulsions, such as incompleteFreund's adjuvant. Other useful adjuvants will be well known to theskilled person.

In yet further aspects, the present invention provides:

(a) the use of OGTA001, an antigenic fragment thereof, or an antigencomposition of the invention in the preparation of an immunogeniccomposition, preferably a vaccine;

(b) the use of such an immunogenic composition in inducing an immuneresponse in a subject; and

(c) a method for the treatment or prophylaxis of colorectal cancer in asubject, or of vaccinating a subject against colorectal cancer whichcomprises the step of administering to the subject an effective amountof OGTA001, at least one antigenic fragment thereof or an antigencomposition of the invention, preferably as a vaccine.

In a specific embodiment, a preparation of OGTA001 or OGTA001 peptidefragments is used as a vaccine for the treatment of colorectal cancer.Such preparations may include adjuvants or other vehicles.

In another embodiment, a preparation of oligonucleotides comprising 10or more consecutive nucleotides complementary to a nucleotide sequenceencoding OGTA001 or OGTA001 peptide fragments is used as vaccines forthe treatment of colorectal cancer. Such preparations may includeadjuvants or other vehicles.

Inhibition of OGTA001 to Treat Colorectal Cancer

In one embodiment of the invention, colorectal cancer is treated orprevented by administration of a compound that antagonizes (inhibits)the level(s) and/or function(s) of OGTA001 which are elevated in theserum or tissue of subjects having colorectal cancer as compared withserum or tissue of subjects free from colorectal cancer.

Compounds useful for this purpose include but are not limited toanti-OGTA001 antibodies (or other affinity reagents such as Affibodies,Nanobodies or Unibodies, and fragments and derivatives containing thebinding region thereof), OGTA001 antisense or ribozyme nucleic acids,and nucleic acids encoding dysfunctional OGTA001 that are used to“knockout” endogenous OGTA001 function by homologous recombination (see,e.g., Capecchi, 1989, Science 244:1288-1292). Other compounds thatinhibit OGTA001 function can be identified by use of known in vitroassays, e.g., assays for the ability of a test compound to inhibitbinding of OGTA001 to another protein or a binding partner, or toinhibit a known OGTA001 function. Preferably such inhibition is assayedin vitro or in cell culture, but genetic assays may also be employed.The Preferred Technologies can also be used to detect levels of OGTA001before and after the administration of the compound. Preferably,suitable in vitro or in vivo assays are utilized to determine the effectof a specific compound and whether its administration is indicated fortreatment of the affected tissue, as described in more detail below.

In a specific embodiment, a compound that inhibits OGTA001 function isadministered therapeutically or prophylactically to a subject in whom anincreased serum or tissue level or functional activity of OGTA001 (e.g.,greater than the normal level or desired level) is detected as comparedwith serum or tissue of subjects free from colorectal cancer or apredetermined reference range. Methods standard in the art can beemployed to measure the increase in OGTA001 level or function, asoutlined above. Preferred OGTA001 inhibitor compositions include smallmolecules, i.e., molecules of 1000 daltons or less. Such small moleculescan be identified by the screening methods described herein.

Assays for Therapeutic or Prophylactic Compounds

The present invention also provides assays for use in drug discovery inorder to identify or verify the efficacy of compounds for treatment orprevention of colorectal cancer. Test compounds can be assayed for theirability to restore OGTA001 levels in a subject having colorectal cancertowards levels found in subjects free from colorectal cancer or toproduce similar changes in experimental animal models of colorectalcancer. Compounds able to restore OGTA001 levels in a subject havingcolorectal cancer towards levels found in subjects free from colorectalcancer or to produce similar changes in experimental animal models ofcolorectal cancer can be used as lead compounds for further drugdiscovery, or used therapeutically. OGTA001 expression can be assayed bythe Preferred Technologies, immunoassays, gel electrophoresis followedby visualization, detection of OGTA001 activity, or any other methodtaught herein or known to those skilled in the art. Such assays can beused to screen candidate drugs, in clinical monitoring or in drugdevelopment, where abundance of OGTA001 can serve as a surrogate markerfor clinical disease.

In various specific embodiments, in vitro assays can be carried out withcells representative of cell types involved in a subject's disorder, todetermine if a compound has a desired effect upon such cell types.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to rats,mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, priorto administration to humans, any animal model system known in the artmay be used. Examples of animal models of colorectal cancer include, butare not limited to xenografts of human colorectal cancer cell lines suchas MDA-MB-435 in oestrogen-deprived Severe Combined Immunodeficient(SCID) mice (Eccles et al., 1994 Cell Biophysics 24/25, 279). These canbe utilized to test compounds that modulate OGTA001 levels, since thepathology exhibited in these models is similar to that of colorectalcancer. It is also apparent to the skilled artisan that based upon thepresent disclosure, transgenic animals can be produced with “knock-out”mutations of the gene or genes encoding OGTA001. A “knock-out” mutationof a gene is a mutation that causes the mutated gene to not beexpressed, or expressed in an aberrant form or at a low level, such thatthe activity associated with the gene product is nearly or entirelyabsent. Preferably, the transgenic animal is a mammal; more preferably,the transgenic animal is a mouse.

In one embodiment, test compounds that modulate the expression ofOGTA001 are identified in non-human animals (e.g., mice, rats, monkeys,rabbits, and guinea pigs), preferably non-human animal models forcolorectal cancer, expressing OGTA001. In accordance with thisembodiment, a test compound or a control compound is administered to theanimals, and the effect of the test compound on expression of OGTA001 isdetermined. A test compound that alters the expression of OGTA001 can beidentified by comparing the level of OGTA001 (or mRNA encoding the same)in an animal or group of animals treated with a test compound with thelevel of OGTA001 or mRNA in an animal or group of animals treated with acontrol compound. Techniques known to those of skill in the art can beused to determine the mRNA and protein levels, for example, in situhybridization. The animals may or may not be sacrificed to assay theeffects of a test compound.

In another embodiment, test compounds that modulate the activity ofOGTA001 or a biologically active portion thereof are identified innon-human animals (e.g., mice, rats, monkeys, rabbits, and guinea pigs),preferably non-human animal models for colorectal cancer, expressingOGTA001. In accordance with this embodiment, a test compound or acontrol compound is administered to the animals, and the effect of atest compound on the activity of OGTA001 is determined. A test compoundthat alters the activity of OGTA001 can be identified by assayinganimals treated with a control compound and animals treated with thetest compound. The activity of OGTA001 can be assessed by detectinginduction of a cellular second messenger of OGTA001 (e.g., intracellularCa²⁺, diacylglycerol, 1P3, etc.), detecting catalytic or enzymaticactivity of OGTA001 or binding partner thereof, detecting the inductionof a reporter gene (e.g., a regulatory element that is responsive toOGTA001 operably linked to a nucleic acid encoding a detectable marker,such as luciferase or green fluorescent protein), or detecting acellular response (e.g., cellular differentiation or cellproliferation). Techniques known to those of skill in the art can beutilized to detect changes in the activity of OGTA001 (see, e.g., U.S.Pat. No. 5,401,639, which is incorporated herein by reference).

In yet another embodiment, test compounds that modulate the level orexpression of OGTA001 are identified in human subjects having colorectalcancer, preferably those having severe colorectal cancer. In accordancewith this embodiment, a test compound or a control compound isadministered to the human subject, and the effect of a test compound onOGTA001 expression is determined by analyzing the expression of OGTA001or the mRNA encoding the same in a biological sample (e.g., serum,plasma, or urine). A test compound that alters the expression of OGTA001can be identified by comparing the level of OGTA001 or mRNA encoding thesame in a subject or group of subjects treated with a control compoundto that in a subject or group of subjects treated with a test compound.Alternatively, alterations in the expression of OGTA001 can beidentified by comparing the level of OGTA001 or mRNA encoding the samein a subject or group of subjects before and after the administration ofa test compound. Techniques known to those of skill in the art can beused to obtain the biological sample and analyze the mRNA or proteinexpression. For example, the Preferred Technologies described herein canbe used to assess changes in the level of OGTA001.

In another embodiment, test compounds that modulate the activity ofOGTA001 are identified in human subjects having colorectal cancer,(preferably those with severe colorectal cancer). In this embodiment, atest compound or a control compound is administered to the humansubject, and the effect of a test compound on the activity of OGTA001 isdetermined. A test compound that alters the activity of OGTA001 can beidentified by comparing biological samples from subjects treated with acontrol compound to samples from subjects treated with the testcompound. Alternatively, alterations in the activity of OGTA001 can beidentified by comparing the activity of OGTA001 in a subject or group ofsubjects before and after the administration of a test compound. Theactivity of OGTA001 can be assessed by detecting in a biological sample(e.g., serum, plasma, or urine) induction of a cellular signaltransduction pathway of OGTA001 (e.g., intracellular Ca²⁺,diacylglycerol, IP3, etc.), catalytic or enzymatic activity of OGTA001or a binding partner thereof, or a cellular response, for example,cellular differentiation, or cell proliferation. Techniques known tothose of skill in the art can be used to detect changes in the inductionof a second messenger of OGTA001 or changes in a cellular response. Forexample, RT-PCR can be used to detect changes in the induction of acellular second messenger.

In a preferred embodiment, a test compound that changes the level orexpression of OGTA001 towards levels detected in control subjects (e.g.,humans free from colorectal cancer) is selected for further testing ortherapeutic use. In another preferred embodiment, a test compound thatchanges the activity of OGTA001 towards the activity found in controlsubjects (e.g., humans free from colorectal cancer) is selected forfurther testing or therapeutic use.

In another embodiment, test compounds that reduce the severity of one ormore symptoms associated with colorectal cancer are identified in humansubjects having colorectal cancer, preferably subjects with severecolorectal cancer. In accordance with this embodiment, a test compoundor a control compound is administered to the subjects, and the effect ofa test compound on one or more symptoms of colorectal cancer isdetermined. A test compound that reduces one or more symptoms can beidentified by comparing the subjects treated with a control compound tothe subjects treated with the test compound. Techniques known tophysicians familiar with colorectal cancer can be used to determinewhether a test compound reduces one or more symptoms associated withcolorectal cancer. For example, a test compound that reduces tumourburden in a subject having colorectal cancer will be beneficial forsubjects having colorectal cancer.

In a preferred embodiment, a test compound that reduces the severity ofone or more symptoms associated with colorectal cancer in a human havingcolorectal cancer is selected for further testing or therapeutic use.

Therapeutic and Prophylactic Compositions and their Use

The invention provides methods of treatment (and prophylaxis) comprisingadministering to a subject an effective amount of a compound of theinvention. In a preferred aspect, the compound is substantially purified(e.g., substantially free from substances that limit its effect orproduce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human. In a specific embodiment, a non-human mammal is thesubject.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid are described above; additionalappropriate formulations and routes of administration are describedbelow.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction can beenteral or parenteral and include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection; intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved, for example, and not by way oflimitation, by local infusion during surgery, topical application, e.g.,by injection, by means of a catheter, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers. In one embodiment,administration can be by direct injection into colorectal tissue or atthe site (or former site) of a malignant tumor or neoplastic orpre-neoplastic tissue.

In another embodiment, the compound can be delivered in a vesicle, inparticular a liposome (see Langer, 1990, Science 249:1527-1533; Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald etal., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med.321:574). In another embodiment, polymeric materials can be used (seeMedical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, New York(1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem.23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989,Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the colon, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E.W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the subject. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment of colorectal cancer can be determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachsubject's circumstances. However, suitable dosage ranges for intravenousadministration are generally about 20-500 micrograms of active compoundper kilogram body weight. Suitable dosage ranges for intranasaladministration are generally about 0.01 pg/kg body weight to 1 mg/kgbody weight. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects (a)approval by the agency of manufacture, use or sale for humanadministration, (b) directions for use, or both.

Determining Abundance of OGTA001 by Imaging Technology

An advantage of determining abundance of OGTA001 by imaging technologymay be that such a method is non-invasive (save that reagents may needto be administered) and there is no need to extract a sample from thesubject.

Suitable imaging technologies include positron emission tomography (PET)and single photon emission computed tomography (SPECT). Visualisation ofOGTA001 using such techniques requires incorporation or binding of asuitable label e.g. a radiotracer such as ¹⁸F, ¹¹C or ¹²³I (see e.g.NeuroRx—The Journal of the American Society for ExperimentalNeuroTherapeutics (2005) 2(2), 348-360 and idem pages 361-371 forfurther details of the techniques). Radiotracers or other labels may beincorporated into OGTA001 by administration to the subject (e.g. byinjection) of a suitably labelled specific ligand. Alternatively theymay be incorporated into a binding affinity reagent (antibody, Affibody,Nanobody, Unibody etc.) specific for OGTA001 which may be administeredto the subject (e.g. by injection). For discussion of use of Affibodiesfor imaging see e.g. Orlova A, Magnusson M, Eriksson T L, Nilsson M,Larsson B, Hoiden-Guthenberg I, Widstrom C, Carlsson J, Tolmachev V,Stahl S, Nilsson F Y, Tumor imaging using a picomolar affinity HER2binding affibody molecule, Cancer Res. 2006 Apr. 15; 66(8):4339-48).

Diagnosis and Treatment of Colorectal Cancer Using Immunohistochemistry

Immunohistochemistry is an excellent detection technique and maytherefore be very useful in the diagnosis and treatment of colorectalcancer. Immunohistochemistry may be used to detect, diagnose, or monitorcolorectal cancer through the localization of OGTA001 antigens in tissuesections by the use of labeled antibodies (or other affinity reagentssuch as Affibodies, Nanobodies or Unibodies), derivatives and analogsthereof, which specifically bind to OGTA001, as specific reagentsthrough antigen-antibody interactions that are visualized by a markersuch as fluorescent dye, enzyme, radioactive element or colloidal gold.

The advancement of monoclonal antibody technology has been of greatsignificance in assuring the place of immunohistochemistry in the modernaccurate microscopic diagnosis of human neoplasms. The identification ofdisseminated neoplastically transformed cells by immunohistochemistryallows for a clearer picture of cancer invasion and metastasis, as wellas the evolution of the tumour cell associated immunophenotype towardsincreased malignancy. Future antineoplastic therapeutical approaches mayinclude a variety of individualized immunotherapies, specific for theparticular immunophenotypical pattern associated with each individualpatient's neoplastic disease. For further discussion see e.g. Bodey B,The significance of immunohistochemistry in the diagnosis and therapy ofneoplasms, Expert Opin Biol Ther. 2002 April; 2(4):371-93.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The prior art documents mentionedherein are incorporated to the fullest extent permitted by law.

Example 1 Identification of Membrane Proteins Expressed in ColorectalCancer Blood and Tissue Samples Using 1D Gel Electrophoresis

Using the following Reference Protocol, membrane proteins extracted fromcolorectal cancer tissue samples were separated by 1D gel and analysed.

1.1 Materials and Methods 1.1.1—Plasma Membrane Fractionation

The cells recovered from the epithelium of a colorectal adenocarcinomawere lysed and submitted to centrifugation at 1000G. The supernatant wastaken, and it was subsequently centrifuged at 3000G. Once again, thesupernatant was taken, and it was then centrifuged at 100 000G.

The resulting pellet was recovered and put on 15-60% sucrose gradient.

A Western blot was used to identify sub cellular markers, and the PlasmaMembrane fractions were pooled.

The pooled solution was either run directly on 1D gels (see section1.1.4 below), or further fractionated into heparin binding andnucleotide binding fractions as described below.

1.1.2—Plasma Membrane Heparin-Binding Fraction

The pooled solution from 1.1.1 above was applied to a Heparin column,eluted from column and run on 1D gels (see section 1.1.4 below).

1.1.3—Plasma Nucleotide-Binding Fraction

The pooled solution from 1.1.1 above was applied to a Cibacrom Blue 3GAcolumn, eluted from column and run on 1D gels (see section 1.1.4 below).

1.1.4-1D Gel Technology Protein or membrane pellets were solubilised in1D sample buffer (1-2 μg/μl). The sample buffer and protein mixture wasthen heated to 95° C. for 3 min.

A 9-16% acrylamide gradient gel was cast with a stacking gel and astacking comb according to the procedure described in Ausubel F. M. etal., eds., 1989, Current Protocols in Molecular Biology, Vol. II, GreenPublishing Associates, Inc., and John Wiley & Sons, Inc., New York,section 10.2, incorporated herein by reference in its entirety.

30-50 micrograms of the protein mixtures obtained from detergent and themolecular weight standards (66, 45, 31, 21, 14 kDa) were added to thestacking gel wells using a 10 microlitre pipette tip and the samples runat 40 mA for 5 hours.

The plates were then prised open, the gel placed in a tray of fixer (10%acetic acid, 40% ethanol, 50% water) and shaken overnight. Followingthis, the gel was primed by 30 minutes shaking in a primer solution(7.5% acetic acid (75 ml), 0.05% SDS (5 ml of 10%)). The gel was thenincubated with a fluorescent dye (7.5% acetic acid, 0.06% OGS in-housedye (600 μl)) with shaking for 3 hrs. Sypro Red (Molecular Probes, Inc.,Eugene, Oreg.) is a suitable dye for this purpose. A preferredfluorescent dye is disclosed in U.S. application Ser. No. 09/412,168,filed on Oct. 5, 1999, which is incorporated herein by reference in itsentirety.

A computer-readable output was produced by imaging the fluorescentlystained gels with an Apollo 3 scanner (Oxford Glycosciences, Oxford,UK). This scanner is developed from the scanner described in WO 96/36882and in the Ph.D. thesis of David A. Basiji, entitled “Development of aHigh-throughput Fluorescence Scanner Employing Internal ReflectionOptics and Phase-sensitive Detection (Total Internal Reflection,Electrophoresis)”, University of Washington (1997), Volume 58/12-B ofDissertation Abstracts International, page 6686, the contents of each ofwhich are incorporated herein by reference. The latest embodiment ofthis instrument includes the following improvements: The gel istransported through the scanner on a precision lead-screw drive system.This is preferable to laying the glass plate on the belt-driven systemthat is defined in the Basiji thesis as it provides a reproducible meansof accurately transporting the gel past the imaging optics.

The gel is secured into the scanner against three alignment stops thatrigidly hold the glass plate in a known position. By doing this inconjunction with the above precision transport system and the fact thatthe gel is bound to the glass plate, the absolute position of the gelcan be predicted and recorded. This ensures that accurate co-ordinatesof each feature on the gel can be communicated to the cutting robot forexcision. This cutting robot has an identical mounting arrangement forthe glass plate to preserve the positional accuracy.

The carrier that holds the gel in place has integral fluorescent markers(Designated M1, M2, M3) that are used to correct the image geometry andare a quality control feature to confirm that the scanning has beenperformed correctly.

The optical components of the system have been inverted. The laser,mirror, waveguide and other optical components are now above the glassplate being scanned. The embodiment of the Basiji thesis has theseunderneath. The glass plate is therefore mounted onto the scanner gelside down, so that the optical path remains through the glass plate. Bydoing this, any particles of gel that may break away from the glassplate will fall onto the base of the instrument rather than into theoptics.

In scanning the gels, they were removed from the stain, rinsed withwater and allowed to air dry briefly and imaged on the Apollo 3. Afterimaging, the gels were sealed in polyethylene bags containing a smallvolume of staining solution, and then stored at 4° C.

Apparent molecular weights were calculated by interpolation from a setof known molecular weight markers run alongside the samples.

1.1.5—Recovery and Analysis of Selected Proteins

Proteins were robotically excised from the gels by the process describedin U.S. Pat. No. 6,064,754, Sections 5.4 and 5.6, 5.7, 5.8 (incorporatedherein by reference), as is applicable to 1D-electrophoresis, withmodification to the robotic cutter as follows: the cutter begins at thetop of the lane, and cuts a gel disc 1.7 mm in diameter from the leftedge of the lane. The cutter then moves 2 mm to the right, and 0.7 mmdown and cuts a further disc. This is then repeated. The cutter thenmoves back to a position directly underneath the first gel cut, butoffset by 2.2 mm downwards, and the pattern of three diagonal cuts arerepeated. This is continued for the whole length of the gel.

NOTE: If the lane is observed to broaden significantly then a correctioncan be made also sideways i.e. instead of returning to a positiondirectly underneath a previous gel cut, the cut can be offset slightlyto the left (on the left of the lane) and/or the right (on the right ofthe lane). The proteins contained within the gel fragments wereprocessed to generate tryptic peptides; partial amino acid sequences ofthese peptides were determined by mass spectroscopy as described inWO98/53323 and application Ser. No. 09/094,996, filed Jun. 15, 1998.

Proteins were processed to generate tryptic digest peptides. Trypticpeptides were analyzed by mass spectrometry using a PerSeptiveBiosystems Voyager—DETM STR Matrix-Assisted Laser Desorption IonizationTime-of-Flight (MALDI-TOF) mass spectrometer, and selected trypticpeptides were analyzed by tandem mass spectrometry (MS/MS) using aMicromass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer(Micromass, Altrincham, U.K.) equipped with a Nanoflow™ electrosprayZ-spray source. For partial amino acid sequencing and identification ofOGTA001, uninterpreted tandem mass spectra of tryptic peptides weresearched using the SEQUEST search program (Eng et al., 1994, J. Am. Soc.Mass Spectrom. 5:976-989), version v.C.1. Criteria for databaseidentification included: the cleavage specificity of trypsin; thedetection of a suite of a, b and y ions in peptides returned from thedatabase, and a mass increment for all Cys residues to account forcarbamidomethylation. The database searched was a database constructedof protein entries in the non-redundant database held by the NationalCentre for Biotechnology Information (NCBI) which is accessible atwww.ncbi.nlm.nih.gov. Following identification of proteins throughspectral-spectral correlation using the SEQUEST program, masses detectedin MALDI-TOF mass spectra were assigned to tryptic digest peptideswithin the proteins identified. In cases where no amino acid sequencescould be identified through searching with uninterpreted MS/MS spectraof tryptic digest peptides using the SEQUEST program, tandem massspectra of the peptides were interpreted manually, using methods knownin the art. (In the case of interpretation of low-energy fragmentationmass spectra of peptide ions see Gaskell et al., 1992, Rapid Commun.Mass Spectrom. 6:658-662).

1.1.6—Discrimination of Colorectal Cancer Associated Proteins

The process to identify OGTA001 uses the peptide sequences obtainedexperimentally by mass spectrometry described above of naturallyoccurring human proteins to identify and organize coding exons in thepublished human genome sequence.

Recent dramatic advances in defining the chemical sequence of the humangenome have led to the near completion of this immense task (Venter, J.C. et al. (2001). The sequence of the human genome. Science 16: 1304-51;International Human Genome Sequencing Consortium. (2001). Initialsequencing and analysis of the human genome Nature 409: 860-921). Thereis little doubt that this sequence information will have a substantialimpact on our understanding of many biological processes, includingmolecular evolution, comparative genomics, pathogenic mechanisms andmolecular medicine. For the full medical value inherent in the sequenceof the human genome to be realised, the genome needs to be ‘organised’and annotated. By this, is meant at least the following three things:(i) The assembly of the sequences of the individual portions of thegenome into a coherent, continuous sequence for each chromosome. (ii)The unambiguous identification of those regions of each chromosome thatcontain genes. (iii) Determination of the fine structure of the genesand the properties of its mRNA and protein products. While thedefinition of a ‘gene’ is an increasingly complex issue (H Pearson: Whatis a gene? Nature (2006) 24: 399-401), what is of immediate interest fordrug discovery and development is a catalogue of those genes that encodefunctional, expressed proteins. A subset of these genes will be involvedin the molecular basis of most if not all pathologies. Therefore animportant and immediate goal for the pharmaceutical industry is toidentify all such genes in the human genome and describe their finestructure.

Processing and Integration of Peptide Masses, Peptide Signatures, ESTsand Public Domain Genomic Sequence Data to Form OGAP® Database

-   -   Discrete genetic units (exons, transcripts and genes) were        identified using the following sequential steps:

-   1. A ‘virtual transcriptome’ is generated, containing the tryptic    peptides which map to the human genome by combining the gene    identifications available from Ensembl and various gene prediction    programs. This also incorporates SNP data (from dbSNP) and all    alternate splicing of gene identifications. Known contaminants were    also added to the virtual transcriptome.

-   2. All tandem spectra in the OGeS Mass Spectrometry Database are    interpreted in order to produce a peptide that can be mapped to one    in the virtual transcriptome. A set of automated spectral    interpretation algorithms were used to produce the peptide    identifications.

-   3. The set of all mass-matched peptides in the OGeS Mass    Spectrometry Database is generated by searching all peptides from    transcripts hit by the tandem peptides using a tolerance based on    the mass accuracy of the mass spectrometer, typically 20 ppm.

-   4. All tandem and mass-matched peptides are combined in the form of    “protein clusters”. This is done using a recursive process which    groups sequences into clusters based on common peptide hits.    Biological sequences are considered to belong to the same cluster if    they share one or more tandem or mass-matched peptide.

-   5. After initial filtering to screen out incorrectly identified    peptides, the resulting clusters are then mapped on the human    genome.

-   6. The protein clusters are then aggregated into regions that define    preliminary gene boundaries using their proximity and the    co-observation of peptides within protein clusters. Proximity is    defined as the peptide being within 80,000 nucleotides on the same    strand of the same chromosome. Various elimination rules, based on    cluster observation scoring and multiple mapping to the genome are    used to refine the output. The resulting ‘confirmed genes’ are those    which best account for the peptides and masses observed by mass    spectrometry in each cluster. Nominal co-ordinates for the gene are    also an output of this stage.

-   7. The best set of transcripts for each confirmed gene are created    from the protein clusters, peptides, ESTs, candidate exons and    molecular weight of the original protein spot.

-   8. Each identified transcript was linked to the sample providing the    observed peptides.

-   9. Use of an application for viewing and mining the data. The result    of steps 1-8 was a database containing genes, each of which    consisted of a number of exons and one or more transcripts. An    application was written to display and search this integrated    genome/proteome data. Any features (OMIM disease locus, InterPro    etc.) that had been mapped to the same Golden Path co-ordinate    system by Ensembl could be cross-referenced to these genes by    coincidence of location and fine structure.

Results

The process was used to generate approximately 1 million peptidesequences to identify protein-coding genes and their exons resulted inthe identification of protein sequences for 18083 genes across 67different tissues and 57 diseases including 506 genes in Bladder cancer,4,713 genes in Breast cancer, 766 genes in Burkitt's lymphoma, 1,371genes in Cervical cancer, 949 genes in Colorectal cancer, 1,782 genes inHepatocellular carcinoma, 2,424 genes in CLL, 978 genes in Lung cancer,1,764 genes in Melanoma, 1,033 genes in Ovarian Cancer, 2,961 genes inPancreatic cancer and 3,307 genes in Prostate cancer, illustrated hereby OGTA001 isolated and identified from colorectal cancer samples.Following comparison of the experimentally determined sequences withsequences in the OGAP® database, OGTA001 showed a high degree ofspecificity to colorectal cancer indicative of the prognostic anddiagnostic nature.

1.2 Results

These experiments identified OGTA001, as further described herein. Thefull-length OGTA001 was detected in the plasma membrane of colorectalcancer samples and was not detected in the cytosol.

FIG. 3 shows the Protein Index for OGTA001. For each gene, the proteinindex uses the mass spectrometry data to assign a score to each disease,relative to the global database. The Protein Index can then be used toidentify cancer specific genes with a high score in cancer indicationsand low/negligible scores in normal and other diseases. The indexcontains ˜1 million peptides sequenced via mass spectrometry from 56diseases. For each gene, this yields a score for each disease andsubcellular location. The results are summarized below:

Protein Index Report for OGTA001 Indications positive: Colorectal cancerDisease controls Acute monocytic leukaemia Acute T-cell leukaemiaAlzheimer's Disease Arthritis Asthma Atherosclerosis B-cellnon-Hodgkin's lymphoma Bladder carcinoma Breast cancer Breast diseases,benign Burkitt's lymphoma Bursitis Cancer, unspecified Cervical cancerChronic lymphocytic leukaemia Chronic obstructive pulmonary diseaseColorectal cancer Dementia, vascular Depression Diabetes and ObesityDiverticulitis Dyslipidaemia Emphysema Focal apocrine metaplasia Gastriccancer Gaucher disease Glioblastoma Hepatoblastoma Hepatocellularcarcinoma Hypertension Lactational foci Leukaemia, unspecified Livercirrhosis Lung cancer Lymphoma, histiocytic Melanoma Metabolic syndromeX Migraine, acute Multiple sclerosis Neuroblastoma Normal ObesityOsteoarthritis Osteosarcoma Ovarian cancer Pancreatic cancer Prostatecancer Prostatic diseases, benign Prostatitis Renal cell cancerRetinoblastoma Schizophrenia Skin ulcer Smoker TeratocarcinomaSubcellular location Birbeck Granules Cell surface digest ChromatinFraction Crude Cell Membrane Cytosol Golgi/Mitochondrial MembraneMembrane Glycoprotein Binding Fraction Mitochondria Nucleus PeroxisomesPlasma Membrane Secreted Soluble Fraction Supernatant Whole Cell

FIG. 3 shows the Protein Index for OGTA001 is very high in colorectalcancer plasma membrane and very low in normal plasma membrane. OGTA001was not detected in any other diseases. This indicates that OGTA001 ispotentially a good marker for colorectal cancer.

Example 2 Identification of Membrane Proteins Expressed in ColorectalCancer Blood and Tissue Samples Using Isotope Tagging For Absolute andRelative Quantitation (iTRAQ)

Using the following Reference Protocol, membrane proteins extracted fromcolorectal cancer tissue and normal adjacent colorectal tissue sampleswere digested, labelled with Isotope Tagging for Absolute & RelativeQuantitation reagents (iTRAQ; Applied Biosystems, Foster City, Calif.,USA) and resulting peptides sequenced by tandem mass spectrometry.

2.1 Materials and Methods 2.1.1—Plasma Membrane Fractionation

The cells recovered from a colorectal cancer or normal adjacent tissuewere lysed and submitted to centrifugation at 1000G. The supernatant wastaken, and it was subsequently centrifuged at 3000G. Once again, thesupernatant was taken, and it was then centrifuged at 100 000G.

The resulting pellet was recovered and put on 15-60% sucrose gradient.

A Western blot was used to identify sub cellular markers, and the PlasmaMembrane fractions were pooled.

The pooled solution was then analysed directly by iTRAQ (see section2.1.2 below).

2.1.2—iTRAQ Methodology

Membrane protein pellets from colorectal cancer and normal adjacenttissue were solubilised in sample buffer (2-4 μg/μl in 0.5% SDS) by theaddition of buffer and then heating to 95° C. for 3 min.

To a volume of each protein solution equating to 50 μg, 150 μl of 0.5Mtriethylammonium bicarbonate (TEAB) solution was added. To each sample,3 μl of 50 mM tris-(2-carboxyethyl)phosphine was added and the mixturewas incubated at 60° C. for 1 hour. 1 μl of cysteine blocking reagent,200 mM methyl methanethiosulphonate (MMTS) in isopropanol, was thenadded. After incubation at room temperature for 10 minutes, 15 μl of 1μg/μl trypsin was added to each sample followed by incubation at 37° C.overnight.

The digested samples were dried under a vacuum and re-constituted with30 μl of 0.5M TEAB solution. 70 μl ethanol was added to each of the fouriTRAQ reagents (114/115/116/117) and one reagent added to each of thefour samples analysed (two colorectal cancer samples and twocorresponding normal adjacent tissue samples) and left at roomtemperature for 1 hour. The specific reagent added to each sample wasrecorded. The four labeled samples were combined & vortexed.

The combined sample was reduced to dryness under a vacuum and de-saltedby loading onto a C 18 spin column, washing with aqueous solvent andthen eluting with 70% acetonitrile. The sample fraction was againreduced to dryness and then re-dissolved in 40 μl of solvent A (97.9water, 2% acetonitrile, 0.1% formic acid) prior to ion exchangefractionation.

2.1.3—Fractionation and Analysis of Labeled Peptides

The sample was fractionated by strong cation exchange chromatographyusing an Agilent 1200 chromatograph (Agilent, Santa Clara, Calif., USA).Samples were eluted off an Agilent Zorbax Bio-SCXII column (3.5 μm;50×0.8 mm) using a 20 μl/min gradient of 0-100 mM sodium acetate over 20minutes and then to 1M over 10 minutes. 1 minute fractions werecollected over the 30 minute run.

Each fraction was analysed by liquid chromatography/mass spectrometryusing an Agilent 1200 chromatograph fitted with a Zorbax 300SB-C18 (150mm×75 μm) and an Agilent 6510 quadrupole—time-of-flight instrument(Agilent, Santa Clara, Calif., USA). Peptides were eluted with a300n1/min gradient increasing from 15% to 45% acetonitrile in 60minutes. Data was acquired in auto MS/MS mode such that up to 3precursor ions above the intensity threshold were selected and production spectra accumulated to facilitate the sequencing of the labeledpeptides. Raw was processed to create peak lists using Spectrum Millsoftware (Agilent, Santa Clara, Calif., USA).

2.1.4—Amino Acid Sequence Analysis of Labeled Peptides

For partial amino acid sequencing and identification of OGTA001,uninterpreted tandem mass spectra of tryptic peptides were searchedusing the SEQUEST search program (Eng et al., 1994, J. Am. Soc. MassSpectrom. 5:976-989). Criteria for database identification included: thecleavage specificity of trypsin; the detection of a suite of a, b and yions in peptides returned from the database, and a mass increment forall cysteine residues to account for modification with methylmethanethiosulphonate and the addition of iTRAQ labels to free amines(N-terminus & lysine). The data was searched through IPI Human v3.23(www.ebi.ac.uk/IPI/IPIhuman.html).

2.1.5—Discrimination of Colorectal Cancer Associated Proteins

The process described in Example 1 section 1.1.6 was employed todiscriminate the colorectal cancer associated proteins in theexperimental samples.

2.2 Results

These experiments identified OGTA001, as further described herein. Thefull-length OGTA001 was detected in the plasma membrane of colorectalcancer samples. The iTRAQ analysis showed that levels of OGTA001 in thecolorectal cancer samples were higher than in the matched normaladjacent tissue samples.

FIG. 2 shows the Protein Index for OGTA001. See Example 1 section 1.2for a description of the Protein Index for OGTA001.

Example 3 Multiplex Assay to Detect Soluble OGTA001 in Patient SerumUsing Luminex Technology

Using the following Reference Protocol, multiplex assays using theLuminex technology were performed using antibodies to soluble OGTA001.

3.1 Materials and Methods

Antibodies to OGTA001 (as defined by SEQ ID No: 1) were developed atBiosite. Each primary antibody to soluble OGTA001 (as defined by SEQ IDNo: 1) was conjugated to a unique Luminex magnetic microsphere (Mugbeads, Luminex Corporation, Austin, Tex.). Mag bead cocktail (50 ul) wasadded to a 96 black well round bottom Costar plate (CorningIncorporated, Corning N.Y.). Using a 96 well magnetic ring stand, theMag beads were pulled down for 1 minute and washed with wash/assaybuffer (PBS with 1% BSA and 0.02% Tween 20). 50 ul of sample or standardwas added along with an additional 50 ul of wash/assay buffer andallowed to incubate on a shaker for 1 hour at room temperature. Platewas placed on magnetic ring stand and allowed to sit for 1 minute. Magbeads were then washed again. Biotin labeled antibody was then added at50 ul per well with an additional 50 ul of wash/assay buffer and allowedto incubate on a shaker for 1 hour at room temperature. The plate againwas placed on a magnetic stand and the Mag beads were washed.Streptavidin-RPE (Prozyme, San Leandro, Calif., Phycolin, Code#PJ31S)was diluted to 1 ug/ml in wash/assay buffer and 50 ul was added to eachwell along with an additional 50 ul of wash/assay buffer and allowed toincubate on a shaker for 1 hour at room temperature. Final wash wasperformed and the beads were re-suspended with 100 ul of wash/assaybuffer and each well was then read in a Luminex 200 reader using Xponentsoftware 3.0. All reagent dilutions were made in wash/assay buffer.Biotin-antibody varied for each assay to optimal concentration. InitialMag bead amounts added were approximately 50,000 for each assay.Magnetic beads were allowed 1 minute pull down time prior to each wash.Each wash step was 3 times washed with 100 ul of wash/assay buffer.Assay standard curves were made in a normal donor patient serum pool.Luminex reader and Mag beads were used and prepared according tomanufacturer guidelines. Standard curves were calculated using a 5parameter log-logistic fit and each sample concentration was determinedfrom this curve fit.

Final Box and ROC results were analyzed using Analyse-it General+Clinical Laboratory 1.73 (Analyse-it Software Ltd., Leeds England).

3.2 Results

Experiments using 61 normal samples and 65 colorectal cancer resulted infurther evidence that soluble OGTA001 can be detected in colorectalcancer patient serum samples and also that the concentration of solubleOGTA001 is higher in colorectal cancer patient serum than in normalserum samples. These results demonstrate that soluble OGTA001 may beused as a serum diagnostic for colorectal cancer.

FIG. 3 shows ROC curve data for soluble OGTA001 in colorectal cancerpatient serum samples. The ROC curves plot sensitivity (true positives)against 1-specificity (false positives). An area under the ROC curve ofgreater than 0.5 indicates good discrimination between disease andnormal. This is the case in the data shown in FIG. 4, which, along withthe low p values, indicate that the concentration of soluble OGTA001 issignificantly higher in colorectal cancer patient serum samples than innormal serum samples.

The following examples and the antibodies prepared and used therein arebased in part, upon disclosure found in co-pending parent InternationalApplication No. PCT/US2010/031739, having an international filing dateof Apr. 20, 2010, and the disclosure of this application is accordinglyincorporated herein by reference in its entirety.

Example 4 Construction of a Phage-Display Library

A recombinant protein composed of the extracellular domain of the CDH17(SEQ ID NO:1) was eurkaryotically synthesized by standard recombinantmethods and used as antigen for immunization.

Immunization and mRNA Isolation

A phage display library for identification of the CDH17-bindingmolecules was constructed as follows. A/J mice (Jackson Laboratories,Bar Harbor, Me.) were immunized intraperitoneally with the recombinantCDH17 antigen (the extracellular domain), using 100 μg protein inFreund's complete adjuvant, on day 0, and with 100 μg antigen on day 28.Test bleeds of mice were obtained through puncture of the retro-orbitalsinus. If, by testing the titers, they were deemed high by ELISA usingthe biotinylated CDH17 antigen immobilized via neutravidin(Reacti-Bind™) NeutrAvidin™-Coated Polystyrene Plates, Pierce, Rockford,Ill.), the mice were boosted with 100 μg of protein on day 70, 71 and72, with subsequent sacrifice and splenectomy on day 77. If titers ofantibody were not deemed satisfactory, mice were boosted with 100 μgantigen on day 56 and a test bleed taken on day 63. If satisfactorytiters were obtained, the animals were boosted with 100 μg of antigen onday 98, 99, and 100 and the spleens harvested on day 105.

The spleens were harvested in a laminar flow hood and transferred to apetri dish, trimming off and discarding fat and connective tissue. Thespleens were macerated quickly with the plunger from a sterile 5 ccsyringe in the presence of 1.0 ml of solution D (25.0 g guanidinethiocyanate (Boehringer Mannheim, Indianapolis, Ind.), 29.3 ml sterilewater, 1.76 ml 0.75 M sodium citrate pH 7.0, 2.64 ml 10% sarkosyl(Fisher Scientific, Pittsburgh, Pa.), 0.36 ml 2-mercaptoethanol (FisherScientific, Pittsburgh, Pa.). This spleen suspension was pulled throughan 18 gauge needle until all cells were lysed and the viscous solutionwas transferred to a microcentrifuge tube. The petri dish was washedwith 100 μl of solution D to recover any remaining spleen. Thissuspension was then pulled through a 22 gauge needle an additional 5-10times.

The sample was divided evenly between two microcentrifuge tubes and thefollowing added, in order, with mixing by inversion after each addition:50 μl 2 M sodium acetate pH 4.0, 0.5 ml water-saturated phenol (FisherScientific, Pittsburgh, Pa.), 100 μl chloroform/isoamyl alcohol 49:1(Fisher Scientific, Pittsburgh, Pa.). The solution was vortexed for 10sec and incubated on ice for 15 min. Following centrifugation at 14 krpmfor 20 min at 2-8° C., the aqueous phase was transferred to a freshtube. An equal volume of water saturated phenol:chloroform:isoamylalcohol (50:49:1) was added, and the tube vortexed for ten seconds.After 15 min incubation on ice, the sample was centrifuged for 20 min at2-8° C., and the aqueous phase transferred to a fresh tube andprecipitated with an equal volume of isopropanol at −20° C. for aminimum of 30 min. Following centrifugation at 14 krpm for 20 min at 4°C., the supernatant was aspirated away, the tubes briefly spun and alltraces of liquid removed from the RNA pellet.

The RNA pellets were each dissolved in 300 μl of solution D, combined,and precipitated with an equal volume of isopropanol at −20° C. for aminimum of 30 min. The sample was centrifuged 14 krpm for 20 min at 4°C., the supernatant aspirated as before, and the sample rinsed with 100μl of ice-cold 70% ethanol. The sample was again centrifuged 14 krpm for20 min at 4° C., the 70% ethanol solution aspirated, and the RNA pelletdried in vacuo. The pellet was resuspended in 100 μl of sterile diethylpyrocarbonate-treated water. The concentration was determined by A260using an absorbance of 1.0 for a concentration of 40 μg/ml. The RNAswere stored at −80° C.

Preparation of Complementary DNA (cDNA)

The total RNA purified from mouse spleens as described above was useddirectly as template for cDNA preparation. RNA (50 μg) was diluted to100 μL with sterile water, and 10 μL of 130 ng/μL oligo dT12(synthesized on Applied Biosystems Model 392 DNA synthesizer) was added.The sample was heated for 10 min at 70° C., then cooled on ice. Forty μL5* first strand buffer was added (Gibco/BRL, Gaithersburg, Md.), alongwith 20 μL 0.1 M dithiothreitol (Gibco/BRL, Gaithersburg, Md.), 10 μL 20mM deoxynucleoside triphosphates (dNTP's, Boehringer Mannheim,Indianapolis, Ind.), and 10 μL water on ice. The sample was thenincubated at 37° C. for 2 min. Ten μL reverse transcriptase(Superscript™) II, Gibco/BRL, Gaithersburg, Md.) was added andincubation was continued at 37° C. for 1 hr. The cDNA products were useddirectly for polymerase chain reaction (PCR).

Amplification of Antibody Genes by PCR

To amplify substantially all of the H and L chain genes using PCR,primers were chosen that corresponded to substantially all publishedsequences. Because the nucleotide sequences of the amino termini of Hand L contain considerable diversity, 33 oligonucleotides weresynthesized to serve as 5′ primers for the H chains, and 29oligonucleotides were synthesized to serve as 5′ primers for the kappa Lchains as described in U.S. Pat. No. 6,555,310. The constant regionnucleotide sequences for each chain required only one 3′ primer for theH chains and one 3′ primer for the kappa L chains.

A 50 μL reaction was performed for each primer pair with 50 μmol of 5′primer, 50 μmol of 3′ primer, 0.25 μL Taq DNA Polymerase (5 units/4,Boehringer Mannheim, Indianapolis, Ind.), 3 μL cDNA (prepared asdescribed), 5 μL 2 mM dNTP's, 5 μL 10*Taq DNA polymerase buffer withMgC12 (Boehringer Mannheim, Indianapolis, Ind.), and H₂O to 50 μL.Amplification was done using a GeneAmp(R) 9600 thermal cycler (PerkinElmer, Foster City, Calif.) with the following thermocycle program: 94°C. for 1 min; 30 cycles of 94° C. for 20 sec, 55° C. for 30 sec, and 72°C. for 30 sec, 72° C. for 6 min; 4° C.

The dsDNA products of the PCR process were then subjected to asymmetricPCR using only a 3′ primer to generate substantially only the anti-sensestrand of the target genes. A 100 μL reaction was done for each dsDNAproduct with 200 μmol of 3′ primer, 2 μL of ds-DNA product, 0.5 μL TaqDNA Polymerase, 10 μL 2 mM dNTP's, 10 μL 10*Taq DNA polymerase bufferwith MgCl₂ (Boehringer Mannheim, Indianapolis, Ind.), and H₂O to 100 μL.The same PCR program as that described above was used to amplify thesingle-stranded (ss)-DNA.

1. Purification of Single-Stranded DNA by High Performance LiquidChromatography and Kinasing Single-Stranded DNA

The H chain ss-PCR products and the L chain single-stranded PCR productswere ethanol precipitated by adding 2.5 volumes ethanol and 0.2 volumes7.5 M ammonium acetate and incubating at −20° C. for at least 30 min.The DNA was pelleted by centrifuging in an Eppendorf centrifuge at 14krpm for 10 min at 2-8° C. The supernatant was carefully aspirated, andthe tubes were briefly spun a 2nd time. The last drop of supernatant wasremoved with a pipette. The DNA was dried in vacuo for 10 min on mediumheat. The H chain products were pooled in 210 μL water and the L chainproducts were pooled separately in 210 μL water. The single-stranded DNAwas purified by high performance liquid chromatography (HPLC) using aHewlett Packard 1090 HPLC and a Gen-Pak™) FAX anion exchange column(Millipore Corp., Milford, Mass.). The gradient used to purify thesingle-stranded DNA is shown in Table 3, and the oven temperature was60° C. Absorbance was monitored at 260 nm. The single-stranded DNAeluted from the HPLC was collected in 0.5 min fractions. Fractionscontaining single-stranded DNA were ethanol precipitated, pelleted anddried as described above. The dried DNA pellets were pooled in 200 μLsterile water.

TABLE 3 HPLC gradient for purification of ss-DNA Time (min) % A % B % CFlow (ml/min) 0 70 30 0 0.75 2 40 60 0 0.75 17 15 85 0 0.75 18 0 100 00.75 23 0 100 0 0.75 24 0 0 100 0.75 28 0 0 100 0.75 29 0 100 0 0.75 340 100 0 0.75 35 70 30 0 0.75 Buffer A is 25 mM Tris, 1 mM EDTA, pH 8.0Buffer B is 25 mM Tris, 1 mM EDTA, 1M NaCl, pH 8.0 Buffer C is 40 mmphosphoric acid

The single-stranded DNA was 5′-phosphorylated in preparation formutagenesis. Twenty-four μL 10* kinase buffer (United StatesBiochemical, Cleveland, Ohio), 10.4 μL 10 mM adenosine-5′-triphosphate(Boehringer Mannheim, Indianapolis, Ind.), and 2 μL polynucleotidekinase (30 units/4, United States Biochemical, Cleveland, Ohio) wasadded to each sample, and the tubes were incubated at 37° C. for 1 hr.The reactions were stopped by incubating the tubes at 70° C. for 10 min.The DNA was purified with one extraction of Tris equilibrated phenol(pH>8.0, United States Biochemical, Cleveland, Ohio):chloroform:isoamylalcohol (50:49:1) and one extraction with chloroform:isoamyl alcohol(49:1). After the extractions, the DNA was ethanol precipitated andpelleted as described above. The DNA pellets were dried, then dissolvedin 50 μL sterile water. The concentration was determined by measuringthe absorbance of an aliquot of the DNA at 260 nm using 33 μg/ml for anabsorbance of 1.0. Samples were stored at −20° C.

2. Preparation of Uracil Templates Used in Generation of Spleen AntibodyPhage Libraries

One ml of E. coli CJ236 (BioRAD, Hercules, Calif.) overnight culture wasadded to 50 ml 2*YT in a 250 ml baffled shake flask. The culture wasgrown at 37° C. to OD600=0.6, inoculated with 10 μl of a 1/100 dilutionof BS45 vector phage stock (described in U.S. Pat. No. 6,555,310) andgrowth continued for 6 hr. Approximately 40 ml of the culture wascentrifuged at 12 krpm for 15 min at 4° C. The supernatant (30 ml) wastransferred to a fresh centrifuge tube and incubated at room temperaturefor 15 min after the addition of 15 μl of 10 mg/ml RNaseA (BoehringerMannheim, Indianapolis, Ind.). The phages were precipitated by theaddition of 7.5 ml of 20% polyethylene glycol 8000 (Fisher Scientific,Pittsburgh, Pa.)/3.5M ammonium acetate (Sigma Chemical Co., St. Louis,Mo.) and incubation on ice for 30 min. The sample was centrifuged at 12krpm for 15 min at 2-8° C. The supernatant was carefully discarded, andthe tube briefly spun to remove all traces of supernatant. The pelletwas resuspended in 400 μl of high salt buffer (300 mM NaCl, 100 mM TrispH 8.0, 1 mM EDTA), and transferred to a 1.5 ml tube.

The phage stock was extracted repeatedly with an equal volume ofequilibrated phenol:chloroform:isoamyl alcohol (50:49:1) until no traceof a white interface was visible, and then extracted with an equalvolume of chloroform:isoamyl alcohol (49:1). The DNA was precipitatedwith 2.5 volumes of ethanol and 1/5 volume 7.5 M ammonium acetate andincubated 30 min at −20° C. The DNA was centrifuged at 14 krpm for 10min at 4° C., the pellet washed once with cold 70% ethanol, and dried invacuo. The uracil template DNA was dissolved in 30 μl sterile water andthe concentration determined by A260 using an absorbance of 1.0 for aconcentration of 40 μg/ml. The template was diluted to 250 ng/μL withsterile water, aliquoted and stored at −20° C.

Mutagenesis of Uracil Template with Ss-DNA and Electroporation into E.Coli to Generate Antibody Phage Libraries

Antibody phage display libraries were generated by simultaneouslyintroducing single-stranded heavy and light chain genes onto a phagedisplay vector uracil template. A typical mutagenesis was performed on a2 μg scale by mixing the following in a 0.2 ml PCR reaction tube: 8 μlof (250 ng/μL) uracil template, 8 μL of 10* annealing buffer (200 mMTris pH 7.0, 20 mM MgCl₂, 500 mM NaCl), 3.33 μl of kinasedsingle-stranded heavy chain insert (100 ng/4), 3.1 μl of kinasedsingle-stranded light chain insert (100 ng/4), and sterile water to 80μl. DNA was annealed in a GeneAmp(R) 9600 thermal cycler using thefollowing thermal profile: 20 sec at 94° C., 85° C. for 60 sec, 85° C.to 55° C. ramp over 30 min, hold at 55° C. for 15 min. The DNA wastransferred to ice after the program finished. The extension/ligationwas carried out by adding 8 μl of 10* synthesis buffer (5 mM each dNTP,10 mM ATP, 100 mM Tris pH 7.4, 50 mM MgCl2, 20 mM DTT), 8 μL T4 DNAligase (1 U/μL, Boehringer Mannheim, Indianapolis, Ind.), 8 μL dilutedT7 DNA polymerase (1 U/μL, New England BioLabs, Beverly, Mass.) andincubating at 37° C. for 30 min. The reaction was stopped with 300 μL ofmutagenesis stop buffer (10 mM Tris pH 8.0, 10 mM EDTA). The mutagenesisDNA was extracted once with equilibrated phenol(pH>8):chloroform:isoamyl alcohol (50:49:1), once withchloroform:isoamyl alcohol (49:1), and the DNA was ethanol precipitatedat −20° C. for at least 30 min. The DNA was pelleted and the supernatantcarefully removed as described above. The sample was briefly spun againand all traces of ethanol removed with a pipetman. The pellet was driedin vacuo. The DNA was resuspended in 4 μL of sterile water.

One μL of mutagenesis DNA (500 ng) was transferred into 40 μlelectrocompetent E. coli DH12S (Gibco/BRL, Gaithersburg, Md.) usingelectroporation. The transformed cells were mixed with approximately 1.0ml of overnight XL-1 cells which were diluted with 2*YT broth to 60% theoriginal volume. This mixture was then transferred to a 15-ml sterileculture tube and 9 ml of top agar added for plating on a 150-mm LB agarplate. Plates were incubated for 4 hr at 37° C. and then transferred to20° C. overnight. First round antibody phage were made by eluting phageoff these plates in 10 ml of 2*YT, spinning out debris, and taking thesupernatant. These samples are the antibody phage display libraries usedfor selecting antibodies against the CDH17. Efficiency of theelectroporations was measured by plating 10 μl of a 10⁻⁴ dilution ofsuspended cells on LB agar plates, follow by overnight incubation ofplates at 37° C. The efficiency was calculated by multiplying the numberof plaques on the 10⁻⁴ dilution plate by 106. Library electroporationefficiencies are typically greater than 1*10⁷ phages under theseconditions.

Transformation of E. Coli by Electroporation

Electrocompetent E. coli cells were thawed on ice. DNA was mixed with 40L of these cells by gently pipetting the cells up and down 2-3 times,being careful not to introduce an air bubble. The cells were transferredto a Gene Pulser cuvette (0.2 cm gap, BioRAD, Hercules, Calif.) that hadbeen cooled on ice, again being careful not to introduce an air bubblein the transfer. The cuvette was placed in the E. coli Pulser (BioRAD,Hercules, Calif.) and electroporated with the voltage set at 1.88 kVaccording to the manufacturer's recommendation. The transformed samplewas immediately resuspended in 1 ml of 2*YT broth or 1 ml of a mixtureof 400 μl 2*YT/600 μl overnight XL-1 cells and processed as proceduresdictated.

Plating M13 Phage or Cells Transformed with Antibody Phage-DisplayVector Mutagenesis Reaction

Phage samples were added to 200 μL of an overnight culture of E. coliXL1-Blue when plating on 100 mm LB agar plates or to 600 μL of overnightcells when plating on 150 mm plates in sterile 15 ml culture tubes.After adding LB top agar (3 ml for 100 mm plates or 9 ml for 150 mmplates, top agar stored at 55° C. (see, Appendix A1, Sambrook et al.,supra.), the mixture was evenly distributed on an LB agar plate that hadbeen pre-warmed (37° C.-55° C.) to remove any excess moisture on theagar surface. The plates were cooled at room temperature until the topagar solidified. The plates were inverted and incubated at 37° C. asindicated.

3. Preparation of Biotinylated Cadherin-17 and Biotinylated Antibodies

The concentrated recombinant CDH17 antigen (full length extracellulardomain SEQ ID No: 67) (Cadherin domains 1-2 SEQ ID No: 68) wasextensively dialyzed into BBS (20 mM borate, 150 mM NaCl, 0.1% NaN₃, pH8.0). After dialysis, 1 mg of the CDH17 (1 mg/ml in BBS) was reactedwith a 15 fold molar excess of biotin-XX-NHS ester (Molecular Probes,Eugene, Oreg., stock solution at 40 mM in DMSO). The reaction wasincubated at room temperature for 90 min and then quenched with taurine(Sigma Chemical Co., St. Louis, Mo.) at a final concentration of 20 mM.The biotinylation reaction mixture was then dialyzed against BBS at 2-8°C. After dialysis, the biotinylated CDH17 was diluted in panning buffer(40 mM Tris, 150 mM NaCl, 20 mg/ml BSA, 0.1% Tween 20, pH 7.5),aliquoted, and stored at −80° C. until needed.

Antibodies were reacted with 3-(N-maleimidylpropionyl)biocytin(Molecular Probes, Eugene, Oreg.) using a free cysteine located at thecarboxy terminus of the heavy chain. Antibodies were reduced by addingDTT to a final concentration of 1 mM for 30 min at room temperature.Reduced antibody was passed through a Sephadex G50 desalting columnequilibrated in 50 mM potassium phosphate, 10 mM boric acid, 150 mMNaCl, pH 7.0. 3-(N-maleimidylpropionyl)-biocytin was added to a finalconcentration of 1 mM and the reaction allowed to proceed at roomtemperature for 60 min. Samples were then dialyzed extensively againstBBS and stored at 2-8° C.

Preparation of Avidin Magnetic Latex

The magnetic latex (Estapor, 10% solids, Bangs Laboratories, Fishers,Ind.) was thoroughly resuspended and 2 ml aliquoted into a 15 ml conicaltube. The magnetic latex was suspended in 12 ml distilled water andseparated from the solution for 10 min using a magnet (PerSeptiveBiosystems, Framingham, Mass.). While maintaining the separation of themagnetic latex with the magnet, the liquid was carefully removed using a10 ml sterile pipette. This washing process was repeated an additionalthree times. After the final wash, the latex was resuspended in 2 ml ofdistilled water. In a separate 50 ml conical tube, 10 mg of avidin-HS(NeutrAvidin, Pierce, Rockford, Ill.) was dissolved in 18 ml of 40 mMTris, 0.15 M sodium chloride, pH 7.5 (TBS). While vortexing, the 2 ml ofwashed magnetic latex was added to the diluted avidin-HS and the mixturemixed an additional 30 sec. This mixture was incubated at 45° C. for 2hr, shaking every 30 min. The avidin magnetic latex was separated fromthe solution using a magnet and washed three times with 20 ml BBS asdescribed above. After the final wash, the latex was resuspended in 10ml BBS and stored at 4° C.

Immediately prior to use, the avidin magnetic latex was equilibrated inpanning buffer (40 mM Tris, 150 mM NaCl, 20 mg/ml BSA, 0.1% Tween 20, pH7.5). The avidin magnetic latex needed for a panning experiment (200μl/sample) was added to a sterile 15 ml centrifuge tube and brought to10 ml with panning buffer. The tube was placed on the magnet for 10 minto separate the latex. The solution was carefully removed with a 10 mlsterile pipette as described above. The magnetic latex was resuspendedin 10 ml of panning buffer to begin the second wash. The magnetic latexwas washed a total of 3 times with panning buffer. After the final wash,the latex was resuspended in panning buffer to the starting volume.

Example 5 Selection of Recombinant Polyclonal Antibodies to CDH17Antigen

Binding reagents that specifically bind to the CDH17 were selected fromthe phage display libraries created from hyperimmunized mice asdescribed in Example 4.

Panning

First round antibody phage were prepared as described in Example 4 usingBS45 uracil template. Electroporations of mutagenesis DNA were performedyielding phage samples derived from different immunized mice. To createmore diversity in the recombinant polyclonal library, each phage samplewas panned separately.

Before the first round of functional panning with the biotinylated CDH17antigen, antibody phage libraries were selected for phage displayingboth heavy and light chains on their surface by panning with7F11-magnetic latex (as described in Examples 21 and 22 of U.S. Pat. No.6,555,310). Functional panning of these enriched libraries was performedin principle as described in Example 16 of U.S. Pat. No. 6,555,310.Specifically, 10 μL of 1*10⁻⁶ M biotinylated CDH17 antigen was added tothe phage samples (approximately 1*10⁻⁸ M final concentration of theCDH17), and the mixture allowed to come to equilibrium overnight at 2-8°C.

After reaching equilibrium, samples were panned with avidin magneticlatex to capture antibody phage bound to the CDH17. Equilibrated avidinmagnetic latex (Example 4), 200 μL latex per sample, was incubated withthe phage for 10 min at room temperature. After 10 min, approximately 9ml of panning buffer was added to each phage sample, and the magneticlatex separated from the solution using a magnet. After a ten minuteseparation, unbound phage was carefully removed using a 10 ml sterilepipette. The magnetic latex was then resuspended in 10 ml of panningbuffer to begin the second wash. The latex was washed a total of threetimes as described above. For each wash, the tubes were in contact withthe magnet for 10 min to separate unbound phage from the magnetic latex.After the third wash, the magnetic latex was resuspended in 1 ml ofpanning buffer and transferred to a 1.5 mL tube. The entire volume ofmagnetic latex for each sample was then collected and resuspended in 200μl 2*YT and plated on 150 mm LB plates as described in Example 1 toamplify bound phage. Plates were incubated at 37° C. for 4 hr, thenovernight at 20° C.

The 150 mm plates used to amplify bound phage were used to generate thenext round of antibody phage. After the overnight incubation, secondround antibody phage were eluted from the 150 mm plates by pipetting 10mL of 2*YT media onto the lawn and gently shaking the plate at roomtemperature for 20 min. The phage samples were then transferred to 15 mldisposable sterile centrifuge tubes with a plug seal cap, and the debrisfrom the LB plate pelleted by centrifuging the tubes for 15 min at 3500rpm. The supernatant containing the second round antibody phage was thentransferred to a new tube.

A second round of functional panning was set up by diluting 100 μL ofeach phage stock into 900 μL of panning buffer in 15 ml disposablesterile centrifuge tubes. The biotinylated CDH17 antigen was then addedto each sample as described for the first round of panning, and thephage samples incubated for 1 hr at room temperature. The phage sampleswere then panned with avidin magnetic latex as described above. Theprogress of panning was monitored at this point by plating aliquots ofeach latex sample on 100 mm LB agar plates to determine the percentageof kappa positives. The majority of latex from each panning (99%) wasplated on 150 mm LB agar plates to amplify the phage bound to the latex.The 100 mm LB agar plates were incubated at 37° C. for 6-7 hr, afterwhich the plates were transferred to room temperature and nitrocellulosefilters (pore size 0.45 mm, BA85 Protran, Schleicher and Schuell, Keene,N.H.) were overlaid onto the plaques.

Plates with nitrocellulose filters were incubated overnight at roomtemperature and then developed with a goat anti-mouse kappa alkalinephosphatase conjugate to determine the percentage of kappa positives asdescribed below. Phage samples with lower percentages (<70%) of kappapositives in the population were subjected to a round of panning with7F11-magnetic latex before performing a third functional round ofpanning overnight at 2-8° C. using the biotinylated CDH17 antigen atapproximately 2*10⁻⁹ M. This round of panning was also monitored forkappa positives. Individual phage samples that had kappa positivepercentages greater than 80% were pooled and subjected to a final roundof panning overnight at 2-8° C. at 5*10⁻⁹ M. The CDH17 antibody genescontained within the eluted phage from this fourth round of functionalpanning were subcloned into the expression vector, pBRncoH3.

The subcloning process was done generally as described in Example 18 ofU.S. Pat. No. 6,555,310. After subcloning, the expression vector waselectroporated into DH10B cells and the mixture grown overnight in 2*YTcontaining 1% glycerol and 10 μg/ml tetracycline. After a second roundof growth and selection in tetracycline, aliquots of cells were frozenat −80° C. as the source for the CDH17 polyclonal antibody production.Monoclonal antibodies were selected from these polyclonal mixtures byplating a sample of the mixture on LB agar plates containing 10 μg/mltetracycline and screening for antibodies that recognized the CDH17.

Expression and Purification of Recombinant Antibodies AgainstCadherin-17

A shake flask inoculum was generated overnight from a −70° C. cell bankin an Innova 4330 incubator shaker (New Brunswick Scientific, Edison,N.J.) set at 37° C., 300 rpm. The inoculum was used to seed a 20 Lfermentor (Applikon, Foster City, Calif.) containing defined culturemedium [Pack et al. (1993) BioTechnology 11: 1271-1277] supplementedwith 3 g/L L-leucine, 3 g/L L-isoleucine, 12 g/L casein digest (Difco,Detroit, Mich.), 12.5 g/L glycerol and 10 μg/ml tetracycline. Thetemperature, pH and dissolved oxygen in the fermentor were controlled at26° C., 6.0-6.8 and 25% saturation, respectively. Foam was controlled byaddition of polypropylene glycol (Dow, Midland, Mich.). Glycerol wasadded to the fermentor in a fed-batch mode. Fab expression was inducedby addition of L(+)-arabinose (Sigma, St. Louis, Mo.) to 2 g/L duringthe late logarithmic growth phase. Cell density was measured by opticaldensity at 600 nm in an UV-1201 spectrophotometer (Shimadzu, Columbia,Md.). Following run termination and adjustment of pH to 6.0, the culturewas passed twice through an M-210B-EH Microfluidizer (Microfluidics,Newton, Mass.) at 17,000 psi. The high pressure homogenization of thecells released the Fab into the culture supernatant.

The first step in purification was expanded bed immobilized metalaffinity chromatography (EB-IMAC). Streamline™ chelating resin(Pharmacia, Piscataway, N.J.) was charged with 0.1 M NiCl₂ and was thenexpanded and equilibrated in 50 mM acetate, 200 mM NaCl, 10 mMimidazole, 0.01% NaN₃, pH 6.0 buffer flowing in the upward direction. Astock solution was used to bring the culture homogenate to 10 mMimidazole, following which it was diluted two-fold or higher inequilibration buffer to reduce the wet solids content to less than 5% byweight. It was then loaded onto the Streamline column flowing in theupward direction at a superficial velocity of 300 cm/hr. The cell debrispassed through unhindered, but the Fab was captured by means of the highaffinity interaction between nickel and the hexahistidine tag on the Fabheavy chain. After washing, the expanded bed was converted to a packedbed and the Fab was eluted with 20 mM borate, 150 mM NaCl, 200 mMimidazole, 0.01% NaN₃, pH 8.0 buffer flowing in the downward direction.

The second step in the purification used ion-exchange chromatography(IEC). Q Sepharose FastFlow resin (Pharmacia, Piscataway, N.J.) wasequilibrated in 20 mM borate, 37.5 mM NaCl, 0.01% NaN₃, pH 8.0. The Fabelution pool from the EB-IMAC step was diluted four-fold in 20 mMborate, 0.01% NaN₃, pH 8.0 and loaded onto the IEC column. Afterwashing, the Fab was eluted with a 37.5-200 mM NaCl salt gradient. Theelution fractions were evaluated for purity using an Xcell II™ SDS-PAGEsystem (Novex, San Diego, Calif.) prior to pooling. Finally, the Fabpool was concentrated and diafiltered into 20 mM borate, 150 mM NaCl,0.01% NaN₃, pH 8.0 buffer for storage. This was achieved in a SartoconSlice™ system fitted with a 10,000 MWCO cassette (Sartorius, Bohemia,N.Y.). The final purification yields were typically 50%. Theconcentration of the purified Fab was measured by UV absorbance at 280nm, assuming an absorbance of 1.6 for a 1 mg/ml solution.

Example 6 Specificity of Monoclonal Antibodies to CDH17 Determined ByFlow Cytometry Analysis

The specificity of antibodies against Cadherin-17 selected in Example 5was tested by flow cytometry. To test the ability of the antibodies tobind to cell surface Cadherin-17 protein, the antibodies were incubatedwith Cadherin-17-expressing cells: LoVo and LS174T, human colorectalcancer lines. Cells were washed and resuspended in PBS. Four microlitersof the suspensions were applied to wells of an eight well microscopeslide and allowed to air dry. The slides were lightly heated to fix thesmears to the slide and covered with 0.1 mg/ml of antibody diluted inPBS containing 1% BSA. The smears were incubated with antibody for 1 hat 37° C. in a moist chamber. After washing the slides three times bysoaking in PBS for 5 min each, the smears were covered with fluoresceinisothiocyanate conjugated rabbit anti-mouse IgG (H&L) F(ab′)2 (ZymedLaboratories, Inc., South San Francisco, Calif.) diluted 1:80 in PBS, 1%BSA, 0.05% Evans Blue (Sigma). The slides were incubated for 1 h at 37°C. in a moist chamber then washed as described above. After a final washin deionized water, the slides were allowed to air dry in the dark.Coverslips were mounted using a 90% glycerol mounting medium containing10 mg/ml p-phenylenediamine, pH 8.0.

4. The results of the flow cytometry analysis demonstrated that 14monoclonal antibodies designated CDH17_A1, CDH17_A2, CDH17_A3, CDH17_A4,CDH17_A5, CDH17_A6, CDH17_A7, CDH17_A8, CDH17_A9, CDH17_A10, CDH17_A11,CDH17_A12, CDH17_A13 and CDH17_A14 bind effectively to cell-surfacehuman Cadherin-17.

Example 7 Structural Characterization of Monoclonal Antibodies ToCadherin-17

The cDNA sequences encoding the heavy and light chain variable regionsof the CDH17_A1, CDH17_A2, CDH17_A3, CDH17_A4, CDH17_A5, CDH17_A6,CDH17_A7, CDH17_A8, CDH17_A9, CDH17_A10, CDH17_A11, CDH17_A12, CDH17_A13and CDH17_A14 monoclonal antibodies were obtained using standard PCRtechniques and were sequenced using standard DNA sequencing techniques.

The antibody sequences may be mutagenized to revert back to germlineresidues at one or more residues.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A1 are SEQ ID NO: 54 and 30, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A1 are SEQ ID NO: 42 and 18, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A2 are SEQ ID NO: 55 and 31, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A2 are SEQ ID NO: 43 and 19, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A3 are SEQ ID NO: 55 and 31, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A3 are SEQ ID NO: 44 and 20, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A4 are SEQ ID NO: 56 and 32, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A4 are SEQ ID NO: 45 and 21, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A5 are SEQ ID NO: 57 and 33, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A5 are SEQ ID NO: 46 and 22, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A6 are SEQ ID NO: 58 and 34, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A6 are SEQ ID NO: 47 and 23, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A7 are SEQ ID NO: 59 and 35, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A7 are SEQ ID NO: 48 and 24, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A8 are SEQ ID NO: 60 and 36, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A8 are SEQ ID NO: 49 and 25, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A9 are SEQ ID NO: 61 and 37, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A9 are SEQ ID NO: 47 and 23, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A10 are SEQ ID NO: 62 and 38, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A10 are SEQ ID NO: 47 and 23, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A11 are SEQ ID NO: 63 and 39, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A11 are SEQ ID NO: 50 and 26, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A12 are SEQ ID NO: 64 and 38, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A12 are SEQ ID NO: 51 and 27, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A13 are SEQ ID NO: 65 and 40, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A13 are SEQ ID NO: 52 and 28, respectively.

The nucleotide and amino acid sequences of the light chain variableregion of CDH17_A14 are SEQ ID NO: 66 and 41, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of CDH17_A14 are SEQ ID NO: 53 and 29, respectively.

Example 8 Immunohistochemistry on FFPE Sections Using Anti-Cadherin-17Antibodies

Immunohistochemistry was performed on FFPE sections of colorectal tumorand normal adjacent tissue using the anti-Cadherin-17 antibodiesCDH17_A3, CDH17_A4, CDH17_A6, CDH17_A8 and CDH17_A9.

EX-De-Wax was from BioGenex, CA, USA. Tissue sections and arrays werefrom Biomax, MD, USA. Slides were heated for 2 h at 60° C. in 50 mlFalcons in a water bath with no buffer. Each Falcon had one slide or twoslides back-to back with long gel loading tip between them to preventslides from sticking to each other. Slides were deparaffinised inEZ-DeWax for 5 min in black slide rack, then rinsed well with the sameDeWax solution using 1 ml pipette, then washed with water from the washbottle. Slides were placed in a coplin jar filled with water until thepressure cooker was ready; the water was changed a couple of times.Water was exchanged for antigen retrieval solution=1×citrate buffer, pH6 (DAKO). Antigen was retrieved by the pressure cooker method. Theslides in the plastic coplin jar in antigen retrieval solution wereplaced into a pressure cooker which was then heated up to position 6(the highest setting). 15-20 min into the incubation, the temperaturewas reduced to position 3 and left at that (when the temperature insidethe pressure cooker was 117° C.) for another 20-25 minutes. Then the hobwas switched off and the cooker was placed onto the cold hob and thepressure was released by carefully moving the handle into the positionbetween “open” and “closed”. The whole system was left to release thepressure and to cool down for another 20 minutes. The lid was opened andsamples taken out to rest on the bench. The slides were washed 1×5 minwith PBS-3T (0.5 L PBS+3 drops of Tween-20) and placed in PBS.

After antigen retrieval, slides were mounted in the Shandon Coverplatesystem. Trapping of air bubbles between the slide and plastic coverplatewas prevented by placing the coverplate into the coplin jar filled withPBS and gently sliding the slide with tissue sections into thecoverplate. The slide was pulled out of the coplin jar while holding ittightly together with the coverplate. The assembled slide was placedinto the rack, letting PBS trapped in the funnel and between the slideand coverplate to run through. Slides were washed with 2×2 ml (or 4×1ml) PBS-3T, 1x2 ml PBS, waiting until all PBS had gone through the slideand virtually no PBS was left in the funnel.

Endogenous peroxide blockade was performed using 1-4 drops of peroxidesolution per slide; the incubation time was 5 minutes. The slides wererinsed with water and then once with 2 ml PBS-3T and once with 2 ml PBS;it was important to wait until virtually no liquid was left in thefunnel before adding a new portion of wash buffer.

The primary antibody was diluted with an Antibody diluent reagent(DAKO). Optimal dilution was determined to be 1:400. Up to 200 μl ofdiluted primary antibody was applied to each slide and incubated for 45minutes at room temperature. Slides were washed with 2×2 ml (or 4×1 ml)PBS-3T and then 1x2 ml PBS.

The goat anti-mouse kappa HRP secondary (1 mg/ml, cat.1050-05, SouthernBiotech) was applied 2×2 drops per slide and incubated for 35 min atroom temperature. The slides were washed as above.

The DAB substrate was made up in dilution buffer; 2 ml containing 2drops of substrate was enough for 10 slides. The DAB reagent was appliedto the slides by applying a few drops at a time and left for 10 min. Theslides were washed 1×2 ml (or 2×1 ml) with PBS-3T and 1x2 ml (or 2×1 ml)with PBS.

Hematoxylin (DAKO) was applied; 1 ml was enough for 10 slides and slideswere incubated for 1 min at room temperature. The funnels of the ShandonCoverplate system were filled with 2 ml of water and let to run through.When slides were clear of the excess of hematoxylin, the system wasdisassembled, tissue sections and/or arrays were washed with water fromthe wash bottle and placed into black slide rack. Tissues weredehydrated by incubating in EZ-DeWax for 5 min and then in 95% ethanolfor 2-5 min.

Slides were left to dry on the bench at room temperature and thenmounted in mounting media and covered with coverslip.

Immunohistochemical analysis on antibodies CDH17_A3, CDH17_A4, CDH17_A6,CDH17_A8 and CDH17_A9 revealed specific membrane staining of tumor cellsin colorectal cancer and no appreciable staining of normal adjacenttissue in all cases. Antibody CDH17_A4, in particular, showed clearspecific membrane staining of tumor cells.

Example 9 Immunohistochemistry on Frozen Sections Using Anti-Cadherin-17Antibodies

Immunohistochemistry was performed on frozen paired tumor and normaladjacent tissues using the anti-Cadherin-17 antibodies CDH17_A4,CDH17_A6, CDH17_A8 and CDH17_A9.

Tissue sections were from BioChain Institute Inc., CA, USA.

Frozen sections were washed with PBS twice for 3 minutes each and werethen placed in PBS.

Endogenous peroxide blockade was performed using Peroxidase Blocker(S2001, DAKO). 1-4 drops of peroxidase blocker was added to each slideand incubated for 5 minutes. The slides were rinsed three times with 3ml PBS.

The primary antibody was diluted with an Antibody diluent reagent(DAKO). 150 μl of diluted primary antibody was applied to each slide andincubated for 45 minutes at room temperature. Slides were washed withtwice for 3 minutes with PBS-3T (500 ml PBS+3 drops of Tween-20) andthen once for 3 minutes with PBS.

The goat anti-mouse kappa HRP secondary was applied at 1:1000 (1 mg/ml,cat.1050-05, Southern Biotech) and incubated for 35 min at roomtemperature. The slides were washed as above.

The DAB substrate was made up in dilution buffer; 2 ml containing 2drops of substrate was enough for 10 slides. The DAB reagent was appliedto the slides by applying a few drops at a time and incubated for 10min. The slides were washed once for 3 minutes with PBS-3T and twice for3 minutes with water.

Hematoxylin (DAKO) was applied; 1 ml was enough for 10 slides and slideswere incubated for 1 min at room temperature.

Slides were left to dry on the bench at room temperature and thenmounted in water-based mounting media from Vector and covered withcoverslip.

Immunohistochemical analysis on antibodies CDH17_A4, CDH17_A6, CDH17_A8and CDH17_A9 on three colorectal cancer samples along with the pairednormal adjacent tissue samples revealed strong specific membranestaining of tumor cells in colorectal cancer and some weak staining ofnormal adjacent tissue. Antibody CDH17_A4, in particular, showed clearspecific membrane staining of tumor cells.

Example 10 Internalization and MabZAP of CDH17_A4 in LS174T and LoVoCells

CDH17_A4 was shown to be internalized by LoVo cells upon binding to thecells using a Immunofluorescence microscopy assay. TheImmunofluorescence microscopy assay showed internalization of theanti-Cadherin-17 monoclonal antibodies through binding of an anti-humanIgG secondary antibody conjugated to Fluorescein isothiocyanate(GamK-FITC). First, CDH17_A4 were bound to the surface of the LoVocells. Then, the secondary antibody conjugated to Fluoresceinisothiocyanate were bound to the primary antibodies. Next, theCDH17_A4/secondary antibody FITC conjugate complex was internalized bythe cells.

The Immunofluorescence microscopy assay was conducted as follows. LoVocell were incubated at 37° C. for 12 hours for cells to adhere to eachother. CDH17_A4 and secondary antibody conjugated to Fluoresceinisothiocyanate were serially diluted, washed with FACS buffer (PBS, 2%FBS) and then added to the culture media. The media was then washedagain with FACS buffer (PBS, 2% FBS) and incubated at 37%, after which200 ul 2% PFA was added. Coverslips were mounted using a 9 ul aqueousmountaing media and the cells were then visualized at regular timeintervals using Leica fluorescent microscope.

The monoclonal antibody, CDH17_A4, was shown to be internalized byLS147T and LoVo cells upon binding to the cells using a MabZap assay.The MabZAP assay showed internalization of the anti-CDH17 monoclonalantibodies through binding of an anti-human IgG secondary antibodyconjugated to the toxin saporin. (Advanced Targeting System, San Diego,Calif., IT-22-100). First, CDH17_A4 was bound to the surface of theLS147T and LoVo cells. Then, the MabZAP antibodies were bound to theprimary antibodies. Next, the MabZAP complex was internalized by thecells. The entrance of Saporin into the cells resulted in proteinsynthesis inhibition and eventual cell death.

The MabZAP assay was conducted as follows. Each of the cells was seededat a density of 5×103 cells per well. The anti-CDH17 monoclonalantibodies or an isotype control human IgG were serially diluted thenadded to the cells. The MabZAP was then added at a concentration of 50μg/ml and the plates allowed too incubate for 48 and 72 hours. Cellviability in the plates was detected by CellTiter-Glo® Luminescent CellViability Assay kit (Promega, G7571) and the plates were read at 490 nMby a Luminomitor (Tuner BioSystems, Sunnyvale, Calif.). The data wasanalyzed by Prism (Graphpad). Cell death was proportional to theconcentration of CDH17_A4 and monoclonal antibody. FIGS. 6A, 6B, 6C, 6D,7A and 7B show that the anti-CDH17 monoclonal antibodies wereefficiently internalized by LS174T and LoVo cells respectively ascompared to the anti-human IgG isotype control antibody.

All references referred to in this application, including patent andpatent applications, are incorporated herein by reference to the fullestextent possible.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

SEQ ID SEQUENCE NO DESCRIPTION SEQUENCE  1 Cadherin-MILQAHLHSLCLLMLYLATGYGQEGKFSGPLKPMTFSIYEGQEPSQIIFQFKANPPAVTFELTGETDNIFVIEREGLL17YYNRALDRETRSTHNLQVAALDANGIIVEGPVPITIEVKDINDNRPTFLQSKYEGSVRQNSRPGKPFLYVNATDLDDPATPNGQLYYQIVIQLPMINNVMYFQINNKTGAISLTREGSQELNPAKNPSYNLVISVKDMGGQSENSFSDTTSVDIIVTENIWKAPKPVEMVENSTDPHPIKITQVRWNDPGAQYSLVDKEKLPRFPFSIDQEGDIYVTQPLDREEKDAYVFYAVAKDEYGKPLSYPLEIHVKVKDINDNPPTCPSPVTVFEVQENERLGNSIGTLTAHDRDEENTANSFLNYRIVEQTPKLPMDGLFLIQTYAGMLQLAKQSLKKQDTPQYNLTIEVSDKDFKTLCFVQINVIDINDQIPIFEKSDYGNLTLAEDTNIGSTILTIQATDADEPFTGSSKILYHIIKGDSEGRLGVDTDPHTNTGYVIIKKPLDFETAAVSNIVFKAENPEPLVFGVKYNASSFAKFTLIVTDVNEAPQFSQHVFQAKVSEDVAIGTKVGNVTAKDPEGLDISYSLRGDTRGWLKIDHVTGEIFSVAPLDREAGSPYRVQVVATEVGGSSLSSVSEFHLILMDVNDNPPRLAKDYTGLFFCHPLSAPGSLIFEATDDDQHLFRGPHFTFSLGSGSLQNDWEVSKINGTHARLSTRHTDFEERAYVVLIRINDGGRPPLEGIVSLPVTFCSCVEGSCFRPAGHQTGIPTVGMAVGILLTTLLVIGIILAVVFIRIKKDKGKDNVESAQASEVKPLRS  2CDH17  AENPEPLVFGVK peptide  3 CDH17  DAYVFYAVAK peptide  4 CDH17 DEENTANSFLNYR peptide  5 CDH17  DEYGKPLSYPLEIHVK peptide  6 CDH17 DINDNRPTFLQSK peptide  7 CDH17  DNVESAQASEVKPLR peptide  8 CDH17 EGSQELNPAK peptide  9 CDH17  GWLK peptide 10 CDH17  IDHVTGEIFSVAPLDRpeptide 11 CDH17  KPLDFETAAVSNIVFK peptide 12 CDH17  LGVDTDPHTNTGYVIIKpeptide 13 CDH17  TGAISLTR peptide 14 CDH17  VKDINDNPPTCPSPVTVFEVQENERpeptide 15 CDH17  VSEDVAIGTK peptide 16 CDH17  WNDPGAQYSLVDK peptide 17CDH17  WNDPGAQYSLVDKEKLPR peptide 18 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAEVQLLETGGGVVKPGGSLKLSCAASGFTFSNYGMSWVacid A1RQTPEKRLEWVAAINRDGGTTYYTDNVKGRFTISRDNAKNSLYLQMSSLRSEDTALYYCARQFLLWDGWYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 19 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPGASVKISCKVSGYTFSDHAIHWMacid A2SQRPGQGLKWIGYIYPRHGTTNYNENFKGKATLTADTSSSTAYMQLNSLTSEDSAVYFCARMRNYFYVMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 20 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVLLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKacid A3QRPEQGLEWIGYIYPEHGTIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCSRLTNYFYVMEYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 21 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAEVQLQQSVAELVKPGASVKMSCKVSGYTLTDHTIHWMacid A4KQRPEQGLEWIGYIYPRDGITGYNEKFKGKATLTADTSSSTAYMQLNSLTSEDSAVYFCARWGYSYRNYAYYYDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 22 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDADLVKPGASVKISCKASGYTFTDHAIHWVacid A5KQRPEQGLEWIGYIYPEHGTIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCARLRNYLYIMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 23 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVacid A6,KQRPEQGLEWIGYIYPEHGTIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCSRLTNYFYVMEYWGQA9, A10GTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 24 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPGASVKISCKVSGYTFTDHAIHWMacid A7KQRPEQGLEWIGYIYPRDGFTKYNEKFKGKATLTADTSSSTAYMQLNSLTSEDSTVYFCARMTNYFYTMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 25 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDADLVKPGASVKISCKASGYTFTDHAIHWVacid A8KQRPEQGLEWIGYIYPEHGTIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCSRLTNYFYVMEYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 26 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVacid A11KQRPEQGLEWIGYIYPEHGSITYNEKFKGKATLTADKSSSTVYMHLNSLTSEDSAVYFCARLRNYLYVMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 27 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSEAELVKPGASVKLSCKASGYTFTDHAIHWMacid A12KQRPEQGLEWIGYIYPRDDFAKVNEKFKGKATLTADTSSSTAYMQLNSLTSEDSAVYFCARMTNYLYIMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 28 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVacid A13KQRPEQGLEWIGYIYPEHGTITYNEKFKGKATLTADKSSSTVYMHLNSLTSEDSAVYFCARLRNYLYIMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 29 VH amino LGKPWRYPRFVHGENKVKQSTIALALLPLLFTPVAKAQVQLQQSDAALVKPGASVKISCKVSGYTFSDHAIHWMacid A14KQRPEQGLEWIGYIFPRDAFSLNNEKFKGKATLSADTSSSTAYMELTSLTFEDSAVYFCARMRNYFYVMDYWGQGTSVTVSSAKTTPPSVYTLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC 30 VK amino RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADVVLTQTPLSLPVTLGDQASISCRSSQSLacid A1LHSNGNTYLHWYLLKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYFCSQSTHVLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 31 VK amino RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCTSSKSLacid A2,LRSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGA3SGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 32 VK amino RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMSQSPSSLAVSVGEKVTMSCKSSQSacid A4LLHSSNQKNYLAWYQQKPGQSPKVLIYWASTRESGVPDRFTGSGSGTDFTLTITSVKSEDLAVYYCQQYYSYPWTFGGGTRLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 33 VK amino RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCRSSKSLacid A5LRSNGNTYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 34 VK amino RILPDAFYRNSLLFLHTRFFGWSETIKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCTSSKSLLacid A6RSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRADAAPTVSILPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 35 VK amino RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCRSSKSLacid A7LRTNGNTYLHWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTVFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 36 VK amino RILPYAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCRSSKSLacid A8LRSNGNTYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 37 VK amino RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCTSSKSLacid A9LRSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRADAAPTVSISPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 38 VK amino RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCRSSKSLacid A10,LRSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGA12GGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 39 VK amino RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVSVTPGESVSISCRSTKSLacid A11LRSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 40 VK amino RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCRSSKSLacid A13LRSNGNTYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRADAAPTVSIFPQYSEQLTTGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNESYPYDVPDYAS 41 VK amino RILPDAFYRNSLLFLHTRFFGWSETMKYLLPTAAAGLLLLAAQPAMADIVMTQAAPSVPVTPGESVSISCTSSKSLacid A14LRSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTNLEIKRADAAPTVSIFTTSREQLTSGGASVVCFLNNFYPKDINVK 42 VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA1ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCGAAGTGCAGCTGTTGGAGACTGGGGGAGGCGTAGTGAAGCCCGGAGGGTCCCTTAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTATGGCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAGCCATTAATCGTGATGGTGGTACCACCTACTATACAGACAATGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACAGCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACAGCCTTGTATTACTGTGCAAGACAGTTCCTTCTCTGGGACGGCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 43VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA2ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGAGCTTCAGTGAAGATATCCTGCAAGGTTTCTGGCTACACCTTCAGTGACCATGCTATTCACTGGATGAGTCAGAGACCTGGACAGGGCCTGAAATGGATTGGATATATTTATCCTAGACATGGGACTACTAACTACAATGAGAACTTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAAGATTCTGCCGTCTATTTCTGTGCAAGAATGAGAAACTACTTCTATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 44 VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA3ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCTGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGAACTATTAAGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCTATATGCAGCTCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTTCAAGACTCACTAACTACTTCTATGTTATGGAGTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 45 VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA4ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCGAGGTTCAGCTGCAGCAGTCTGTCGCTGAGTTGGTGAAACCTGGAGCTTCAGTGAAGATGTCATGCAAGGTTTCTGGCTACACCCTCACTGACCATACTATTCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTACCCTAGAGATGGAATAACTGGGTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACACTTCTTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTCTATTTCTGTGCCAGATGGGGCTATAGTTACAGGAATTACGCGTACTACTATGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 46VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA5ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGACTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGAACTATTAAGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCTATATGCAGCTCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTGCAAGACTCAGGAACTATTTGTATATTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 47 VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA6, A9, ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGAA10GTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGAACTATTAAGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCTATATGCAGCTCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTTCAAGACTCACTAACTACTTCTATGTTATGGAGTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 48 VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA7ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGAGCCTCAGTGAAGATATCCTGCAAGGTTTCTGGCTACACCTTCACTGACCATGCTATTCACTGGATGAAACAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTAGAGATGGTTTTACTAAGTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTACAGTCTATTTCTGTGCAAGAATGACTAACTACTTCTATACTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 49 VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA8ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGACTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGAACTATTAAGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACTGCCTATATGCAGCTCAACAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTTCAAGACTCACTAACTACTTCTATGTTATGGAGTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 50 VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA11ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGTAGTATTACGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGTACTGTCTATATGCACCTCAATAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTGCAAGACTCAGGAACTACTTGTATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 51  VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA12ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGAGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGGATGAAACAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATCTACCCCAGAGATGATTTTGCTAAGGTGAATGAGAAGTTCAAGGGCAAGGCCACACTGACAGCAGACACATCCTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTCTATTTCTGTGCAAGAATGACTAACTACCTCTATATTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 52  VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA13ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTATCCTGAACATGGTACTATTACGTATAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGTACTGTCTATATGCACCTCAATAGCCTGACATCTGAGGATTCAGCAGTGTATTTCTGTGCAAGACTCAGGAACTATTTGTATATTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCCGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 53  VH n.t. TGACTGGGAAAACCCTGGCGTTACCCACGCTTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCA14ACTGGCACTCTTACCGCTCTTATTTACCCCTGTGGCAAAAGCCCAGGTTCAGCTGCAACAGTCTGACGCCGCGTTGGTGAAACCTGGAGCTTCAGTGAAGATATCGTGCAAGGTTTCTGGCTACACCTTCAGTGACCATGCTATTCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATATTTTTCCTAGAGATGCTTTTAGTTTGAACAATGAGAAGTTCAAGGGCAAGGCCACACTGAGTGCAGACACATCCTCCAGCACAGCCTACATGGAGCTCACCAGCCTGACATTTGAGGATTCTGCAGTCTATTTCTGTGCAAGAATGAGAAACTACTTCTATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATACACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT 54  VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA1GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATGTTGTGCTGACCCAGACTCCACTCTCCCTGCCTGTCACTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTTTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCTGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCACCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 55  VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA2, A3GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 56  VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA4GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGACATCGTTATGTCTCAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGCCAGAGCCTTTTACATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAAGTGCTGATTTACTGGGCATCCACTAGAGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACCAGTGTGAAGTCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCGTGGACGTTCGGTGGCGGCACCAGGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 57  VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA5GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCGCAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGCTGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCTGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACATTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 58  VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA6GAGTGAAACGATAAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCCTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 59  VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA7GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTTTCCATCTCCTGCAGGTCTTCTAAGAGTCTCCTGCGTACTAATGGCAACACTTACTTGCATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGTTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAAGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 60  VK n.t. TAAGATTAGCGGATCCTACCTTACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA8GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAATCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGCTGTCTAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACATTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 61  VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA9GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTCCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 62  VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA10GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCCAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTCATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCCTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 63 VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTTCTGTTTCTCCATACCCGTTTTTTTGGATGA11GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTATCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTACTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTCCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 64 VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA12GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCCTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 65 VK n.t. TAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGA13GAGTGAAACGATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCGCAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGCTGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACATTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACAATACAGTGAGCAGTTAACAACTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTCTTATCCATATGATGTGCCAGATTATGCGAGCTAA 66 VK n.t. CGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATGGAGTGAAACA14GATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCCATGGCCGATATTGTGATGACCCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCACGTCTAGTAAGAGTCTCCTGCGTAGTAATGGCAACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCGGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTTCGGCTCGGGGACAAATTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCACAACATCCAGAGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAG 67 Cadherin-17 QEGKFSGPLKPMTFSIYEGQEPSQIIFQFKANPPAVTFELTGETDNIFVIEREGLLYYNRALDRETRSTHNLQVAALECDDANGIIVEGPVPITIKVKDINDNRPTFLQSKYEGSVRQNSRPGKPFLYVNATDLDDPATPNGQLYYQIVIQLPMINNVMYFQINNKTGAISLTREGSQELNPAKNPSYNLVISVKDMGGQSENSFSDTTSVDIIVTENIWKAPKPVEMVENSTDPHPIKITQVRWNDPGAQYSLVDKEKLPRFPFSIDQEGDIYVTQPLDREEKDAYVFYAVAKDEYGKPLSYPLEIHVKVKDINDNPPTCPSPVTVFEVQENERLGNSIGTLTAHDRDEENTANSFLNYRIVEQTPKLPMDGLFLIQTYAGMLQLAKQSLKKQDTPQYNLTIEVSDKDFKTLCFVQINVIDINDQIPIFEKSDYGNLTLAEDTNIGSTILTIQATDADEPFTGSSKILYHIIKGDSEGRLGVDTDPHTNTGYVIIKKPLDFETAAVSNIVFKAENPEPLVFGVKYNASSFAKFTLIVTDVNEAPQFSQHVFQAKVSEDVAIGTKVGNVTAKDPEGLDISYSLRGDTRGWLKIDHVTGEIFSVAPLDREAGSPYRVQVVATEVGGSSLSSVSEFHLILMDVNDNPPRLAKDYTGLFFCHPLSAPGSLIFEATDDDQHLFRGPHFTFSLGSGSLQNDWEVSKINGTHARLSTRHTDFEEREYVVLIRINDGGRPPLEGIVSLPVTFCSCVEGSCFRPAGHQTGIPTVGM68 Cadherin-QEGKFSGPLKPMTFSIYEGQEPSQIIFQFKANPPAVTFELTGETDNIFVIEREGLLYYNRALDRETRSTHNLQVAAL17 ECDDANGIIVEGPVPITIKVKDINDNRPTFLQSKYEGSVRQNSRPGKPFLYVNATDLDDPATPNGQLYYQIVIQLPMINNdomains 1-2VMYFQINNKTGAISLTREGSQELNPAKNPSYNLVISVKDMGGQSENSFSDTTSVDIIVTENIWKAPKP

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.(canceled)
 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)16. (canceled)
 17. (canceled)
 18. A method for screening for and/ordiagnosis of colorectal cancer in a human subject, which comprises thestep of identifying the presence or absence of Cadherin-17, or afragment thereof, in a biological sample obtained from said humansubject, wherein the method comprises an immunoassay step utilising oneor more antibodies or other affinity reagents such as Affibodies,Nanobodies or Unibodies against Cadherin-17 or a fragment or derivativethereof.
 19. A method for monitoring and/or assessing colorectal cancertreatment in a human subject, which comprises the step of identifyingthe presence or absence of Cadherin-17, or a fragment thereof, in abiological sample obtained from said human subject, wherein the methodcomprises an immunoassay step utilising one or more antibodies or otheraffinity reagents such as Affibodies Nanobodies or Unibodies againstCadherin-17 or a fragment or derivative thereof.
 20. A method foridentifying the presence or absence of metastatic colorectal cancercells in a biological sample obtained from a human subject, whichcomprises the step of identifying the presence or absence ofCadherin-17, or a fragment thereof, wherein the method comprises animmunoassay step utilising one or more antibodies or other affinityreagents such as Affibodies, Nanobodies or Unibodies against Cadherin-17or a fragment or derivative thereof.
 21. (canceled)
 22. (canceled) 23.(canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)28. (canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled) 32.(canceled)
 33. (canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled)37. (canceled)
 38. (canceled)
 39. (canceled)
 40. (canceled) 41.(canceled)
 42. (canceled)
 43. (canceled)
 44. (canceled)
 45. (canceled)46. (canceled)
 47. (canceled)
 48. (canceled)
 49. (canceled) 50.(canceled)
 51. (canceled)
 52. (canceled)
 53. (canceled)
 54. (canceled)55. (canceled)
 56. (canceled)
 57. (canceled)
 58. (canceled) 59.(canceled)
 60. (canceled)
 61. A method as claimed in claim 18, whereinthe antibody is selected from the group consisting of polyclonal,monoclonal, bispecific, humanized, chimeric and single chain antibodies,Fab fragments and F(ab′)₂ fragments.
 62. A method as claimed in claim19, wherein the antibody is selected from the group consisting ofpolyclonal, monoclonal, bispecific, humanized, chimeric and single chainantibodies, Fab fragments and F(ab′)₂ fragments.
 63. A method as claimedin claim 20, wherein the antibody is selected from the group consistingof polyclonal, monoclonal, bispecific, humanized, chimeric and singlechain antibodies, Fab fragments and F(ab′)₂ fragments.
 64. A method asclaimed in claim 18, wherein the antibody or affinity reagent isconjugated to a diagnostic moiety.
 65. A method as claimed in claim 19,wherein the antibody or affinity reagent is conjugated to a diagnosticmoiety.
 66. A method as claimed in claim 20, wherein the antibody oraffinity reagent is conjugated to a diagnostic moiety.
 67. A method asclaimed in claim 61, wherein the antibody is conjugated to a diagnosticmoiety.
 68. A method as claimed in claim 62, wherein the antibody isconjugated to a diagnostic moiety.
 69. A method as claimed in claim 63,wherein the antibody is conjugated to a diagnostic moiety.